ultrafiltration handbook Tools and Techniques for Life Science Research

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1 Â ultrafiltration handbook Tools and Techniques for Life Science Research

2 Introduction This handbook is written for research scientists who are using, or who are interested in using, ultrafiltration (UF) technology in their work. Our goal is to provide researchers with a handy reference piece that will allow them to optimize their UF separations and provide them with purified and/or concentrated sample that is ready for downstream experimentation. The intention is not to make ultrafiltration experts out of research scientists in fact, one of the appealing benefits of the technology is its simplicity. However, having a basic understanding of how ultrafiltration works, and how it can be applied to everyday research, enables the researcher to select the right membrane or device for their particular experiment. The information contained in this handbook is culled from decades of collective experience of Millipore and Amicon applications scientists and technical service employees, as well as the company s library of technical publications on ultrafiltration, UF devices, and UF applications. Millipore customers have also contributed data, helping to make this handbook a complete resource for ultrafiltration. Ultrafiltration has evolved significantly since it was first introduced in the 1970s, and it will continue to evolve as life science research progresses. New device platforms and new applications work have expanded the use of UF into new areas, such as genomics, proteomics, drug compound screening, and small molecule assays. To keep up with the latest developments, visit our web site: The Protein Research Team Millipore Corporation Life Sciences Division

3 Table of Contents Overview of Membrane Filtration Membrane Processes Ultrafiltration Microfiltration Reverse Osmosis Recovery Nominal Molecular Weight Limit Nucleotide Cut-off Retention Concentration Polarization Flux Effects of Operating Parameters on Flux...6 Importance of Recovery Mode of Operation Normal vs. Tangential Flow Filtration Diafiltration Dialysis vs. Diafiltration Guide to Selecting Membranes and Devices Membrane Selection Types of Membranes Membranes Organized by Typical Recoveries Membranes Organized by Application..10 Device Selection Small Volume Devices Medium Volume Devices Large Volume and TFF Devices Stirred Cells Specialty Devices and Kits Protocols Proteins Concentration, Desalting, and Buffer Exchange with Amicon Ultra or Microcon Centrifugal Filters Detergent Removal with Microcon, Amicon Ultra or Centricon Centrifugal Filters Two-Dimensional Electrophoresis Sample Preparation with Amicon Ultra Centrifugal Filters Purification of Serum Peptides for Biomarker Research with Amicon Ultra Centrifugal Filters Rapid Antibody Concentration with Amicon Ultra Centrifugal Filters Removal of Unincorporated Label from Labeled Protein with Amicon Ultra or Centricon Centrifugal Filters Urine Concentration with Amicon Ultra Centrifugal Filters Affinity Purification with Ultrafree-MC Centrifugal Filters Use of Centrifugal Filter Devices as an Alternative to Stirred Cells Nucleic Acids Concentrating and Desalting DNA or RNA with Microcon or Centricon Centrifugal Filters Preparing Samples for Forensics Identification Analysis with Microcon Centrifugal Filters PCR Purification with Montage PCR Filter Units Preparation of Fluorescent DNA Probe from Human mrna or Total RNA with Microcon Centrifugal Filters Purification of In Vitro Synthesized mrna with Microcon or Centricon Centrifugal Filters Quantitative Recoveries of Nanogram Amounts of Nucleic Acids with Microcon Centrifugal Filters Effect of Centrifugal Ultrafiltration on Large Fragment DNA Integrity DNA Extraction from Agarose Gels with Montage Gel Extraction Kit or Ultrafree-DA Centrifugal Filters RNA Purification with Microcon or Centricon Centrifugal Filters Enzyme Removal with Micropure-EZ Centrifugal Filters High Throughput Applications Using MultiScreen Filter Plates with Ultracel-10 Ultrafiltration Membrane..58 Appendix Glossary

4 Overview of Membrane Filtration Membrane Processes Ultrafiltration Ultrafiltration (UF) is the process of separating extremely small particles and dissolved molecules from fluids. The primary basis for separation is molecular size, although in all filtration applications, the permeability of a filter medium can be affected by the chemical, molecular or electrostatic properties of the sample. Ultrafiltration can only separate molecules which differ by at least an order of magnitude in size. Molecules of similar size can not be separated by ultrafiltration (see Figure 1.3). Materials ranging in size from 1K to 1000K molecular weight (MW) are retained by certain ultrafiltration membranes, while salts and water will pass through. Colloidal and particulate matter can also be retained. Ultrafiltration membranes can be used both to purify material passing through the filter and also to collect material retained by the filter. Materials significantly smaller than the pore size rating pass through the filter and can be depyrogenated, clarified and separated from high molecular weight contaminants. Materials larger than the pore size rating are retained by the filter and can be concentrated or separated from low molecular weight contaminants. Ultrafiltration is typically used to separate proteins from buffer components for buffer exchange, desalting, or concentration. Ultrafilters are also ideal for removal or exchange of sugars, non-aqueous solvents, the separation of free from protein-bound ligands, the removal of materials of low molecular weight, or the rapid change of ionic and/or ph environment (see Figure 1.6, page 8). Depending on the protein to be retained, the most frequently used membranes have a nominal molecular weight limit (NMWL) of 3 kda to 100 kda. Ultrafiltration is far gentler to solutes than processes such as precipitation. UF is more efficient because it can simultaneously concentrate and desalt solutes. It does not require a phase change, which often denatures labile species, and UF can be performed either at room temperature or in a cold room. Microfiltration Microfiltration (MF) is the process of removing particles or biological entities in the µm to 10.0 µm range from fluids by passage through a microporous medium such as a membrane filter. Although micron-sized particles can be removed by use of non-membrane or depth materials such as Reverse Osmosis Ultrafiltration Microfiltration Clarification µm µm 0.01 µm 0.1 µm 1 µm 10 µm 100 µm 0.2 kda 200 kda 20,000 kda Sugars Proteins B. diminuta Red Blood Cell Smallest Visible Particle Amino Acids Nucleotides Carbon Black Yeast Pollens Salts Oligonucleotides Polio Virus Mycoplasma E. coli Clouds Fog Antibiotics Mammalian Virus Bacteria Human Hair Figure 1.1 Comparison of ultrafiltration with other commonly used membrane separation techniques. 2

5 those found in fibrous media, only a membrane filter having a precisely defined pore size can ensure quantitative retention. Membrane filters can be used for final filtration or prefiltration, whereas a depth filter is generally used in clarifying applications where quantitative retention is not required or as a prefilter to prolong the life of a downstream membrane. Membrane and depth filters offer certain advantages and limitations. They can complement each other when used together in a microfiltration process system or fabricated device. The retention boundary defined by a membrane filter can also be used as an analytical tool to validate the integrity and efficiency of a system. For example, in addition to clarifying or sterilizing filtration, fluids containing bacteria can be filtered to trap the microorganisms on the membrane surface for subsequent culture and analysis. Microfiltration can also be used in sample preparation to remove intact cells and some cell debris from the lysate. Membrane pore size cut-offs used for this type of separation are typically in the range of 0.05 µm to 1.0 µm. Figure 1.2 Ultrafiltration membranes vs. traditional microporous membranes Cross-section of ultrafiltration membrane with skin and porous substructure. Ultrafiltration membranes generally have two distinct layers: a thin ( µm), dense skin with a pore diameter of Å and a more porous substructure. Any species capable of passing through the pores of the skin (whose size is precisely controlled in manufacture) can therefore freely pass the membrane. Cross-section of traditional microporous membrane with uniform pore structure from top to bottom. Microporous membranes are generally rigid, continuous meshes of polymeric material with defined pore sizes. They are used to retain bacteria, colloids and particulates. Species are either retained on the membrane surface or trapped in its substructure. Figure 1.3 Fractionating samples using ultrafiltration membranes A popular misconception is that one can pass a mixture of macromolecules through an ultrafiltration membrane and separate all molecules whose molecular weights are larger than the membrane s cut-off from those whose molecular weights are smaller than the cut-off. This expectation is based on a misunderstanding of the principles of membrane filtration. Most scientists believe that molecules smaller than the membrane rating will pass freely through to the filtrate while molecules larger than the membrane rating will be quantitatively retained. In fact, membrane ratings are based on retention and not on passage properties. The relative retention/passage properties for a 100K NMWL rated membrane are illustrated below. These data were generated using Millipore s mixed dextran rejection procedure. This protocol provides a more detailed look at membrane retention Rejection Coefficient Ultracel PL 300K NMWL membrane , ,000 1,000,000 Molecular Weight (kda) Mixed Dextran Rejection Profile properties than the traditional evaluation with two or three protein species. The mixed dextran protocol uses a total of 11 dextran standards of known molecular weight (180 to 1750K daltons) and the relative retention/passage of the mixture is determined after filtration by analyzing the retentate and filtrate fractions using gel permeation chromatography. An examination of the curve for the Ultracel PL 300K NMWL membrane confirms that molecules larger that the 100K NMWL rating are retained at a level greater than 90%. However, molecules smaller than the membrane rating do not exhibit free passage to the filtrate; for example, a 75K kda species is retained at a 83% level and a 30K kda species is 32% retained on this membrane. These smaller molecules are being retained by a membrane with a higher retention rating because the retention/passage phenomenon is a function of both the membrane and the solution being processed. Buffer composition as well as solute concentration can have major impacts on the retention/passage of molecules. For example, dilute solutions of single solutes less than the NMWL ratings are more likely to appear in the filtrate than the same molecule in a more concentrated solution or in a complex mixture. However, it may be possible to affect this type of separation between two macromolecules if there is at least 10X difference in their molecular weight and if there is an ultrafiltration membrane available with a NMWL rating between the molecular weights of the two proteins. These separations are also generally more successful if the protein solution is dilute and the separation is run at low pressure. 3

6 Reverse Osmosis Reverse osmosis (RO) separates salts and small molecules from low molecular weight solutes (typically less than 100 daltons) at relatively high pressures using membranes with NMWLs of 1 kda or lower. RO membranes are normally rated by their retention of sodium chloride while ultrafiltration membranes are characterized according to the molecular weight of retained solutes. Millipore water purification systems employ both reverse osmosis membranes as well as ultrafiltration membranes. Reverse osmosis systems are primarily used to purify tap water to purities that exceed distilled water quality. Ultrafiltration systems ensure that ultrapure water is free from endotoxins as well as nucleases for critical biological research. Recovery The ultimate aim of ultrafiltration is to maximize recovery of solutes of interest, but there are many membrane characteristics that affect that goal. Factors affecting recovery include: Nominal molecular weight limit (NMWL)/ nucleotide cut-off (NCO) Retention Concentration polarization Flux Nominal Molecular Weight Limit A microfiltration membrane s pore size rating, typically given as a micron value, indicates that particles larger than the rating will be retained. Ultrafiltration membranes are rated according to the nominal molecular weight limit (NMWL), also sometimes referred to as molecular weight cut-off (MWCO). The NMWL indicates that most dissolved macromolecules with molecular weights higher than the NMWL will be retained. An ultrafiltration membrane with a stated NMWL should retain (reject) at least 90% of a globular solute of that molecular weight in daltons. However, for a wider safety margin, the selected cut-off should be well below the molecular weight of the solute to be retained. When solutes are to be exchanged, the cut-off should be substantially above that of the passing solute. A lower NMWL increases rejection but decreases the filtration rate for the same membrane material. Retention and product recovery are a function of a variety of other factors, including the molecular shape and size of the molecule; electrical characteristics; sample concentration and composition; operating conditions; and device or system configuration. Two membranes may have the same NMWL but will exhibit different retention of molecules within a relatively narrow range of sizes. In addition, slender, linear molecules (e.g., nucleic acids) may find their way through pores that will retain a globular species of the same weight. Retention can also be affected by hydration with counter-ions. Nevertheless, NMWL has proven to be an effective general indicator of membrane performance for globular proteins. When using membrane ultrafiltration for sample concentration or desalting, care must be taken to select a membrane (or device) with a NMWL appropriate for the application. Because there are several considerations in determining whether a given solute will or will not be retained by a membrane of a specific cut-off, it is best to choose a device with cut-off at about one half of the molecular weight of the protein to be concentrated. This maximizes protein recovery and minimizes filtration time. Nucleotide Cut-off (NCO) For most membranes, the NMWL is determined experimentally under a standard set of operating conditions. These analyses typically employ purified globular proteins to serve as markers or indicators of the retention characteristics of an ultrafiltration membrane. Although this approach is useful for choosing the appropriate NMWL for most protein research applications, selection of a membrane with an appropriate NMWL membrane for nucleic acid or polysaccharide purification is considerably more complex. By virtue of the rod-like three-dimensional structures of these molecules, these types of molecules require a tighter membrane (with a smaller cut-off) than do globular proteins of the same molecular weight. It is therefore convenient to consider the membrane retention characteristics of nucleic acids as being related to their length (in nucleotides) rather than their molecular weight. Complicating matters even further are several additional factors that affect the recovery of nucleic acid fragments from a membrane of a given NMWL. These factors include: the strandedness of the DNA or RNA molecule; whether the DNA is linear, relaxed or supercoiled (for plasmid); the ionic strength of the solvent; the velocity of the process stream over the membrane; and the nature of the driving force. The overall effect is that optimal 4

7 nucleic acid recovery is achieved in low salt buffers run under conditions of relatively low velocity (e.g., low vacuum pressure or low g-force). The membrane and protocol developed for the Montage PCR centrifugal filter device takes into account these conditions in order to provide for high recovery of small PCR products (e.g., ~150 base pairs [bp]) as quickly as possible. For purifications that are driven by vacuum, significantly tighter membranes are typically required to obtain optimal recovery. If the DNA sample is in the presence of high salt (or the device is run at a higher-than-recommended g-force), a significantly reduced DNA recovery may be observed. Under these conditions, higher DNA or RNA recovery can be achieved by using a tighter membrane. However, it will take significantly longer to complete the purification. For applications such as PCR where removal of unincorporated singlestranded primers from double-stranded DNA fragments is required, the molecular weights of the primer and DNA fragment should differ by at least an order of magnitude for efficient separation. Millipore offers devices that are specifically designed for separating and concentrating genomic DNA and PCR products by ultrafiltration. Retention Retention, also sometimes called rejection, is a function of molecular size and shape. Nominal cutoff levels, defined with model solutes, are convenient indicators. Degree of hydration, counter ions, and steric effects can cause molecules with similar molecular weights to exhibit very different retention behavior. Many biological macromolecules tend to aggregate, or change conformation under varying conditions of ph and ionic strength, so that effective size may be much larger than the native molecule, causing increased rejection. Solute/solvent and solute/solute interactions in the sample can also change effective molecular size. For example, some proteins will polymerize under certain concentration and buffer conditions while others (e.g., heme proteins) may break into corresponding subunits. Ionic interactions or π π stacking can cause small molecules to behave similarly to molecules of greater molecular weight. When this occurs, as in the case of phosphate ions with a 500 NMWL membrane, the small molecules may not effectively permeate the membrane. Millipore recommends the selection of a membrane filter NMWL that is one half the size of the molecule of interest. Other manufacturers may recommend a smaller differential between the size of the NMWL and the size of the molecule but Millipore s recommendation is designed to provide maximum recovery. Please see additional information regarding membrane NMWL selection on page 4. Concentration Polarization Another factor affecting the retention characteristics is the potential for membrane fouling, or concentration polarization. This occurs when there is an accumulation of the retained solute on the surface of the membrane. At high concentrations, a gel layer forms that can act as a secondary membrane (Figure 1.4). This may interfere with passage of the molecules through the membrane and can adversely affect the flow rate. In addition, ph, buffer components, and concentration can result in a protein behaving in an anomalous manner in terms of its retention or passage by UF membranes. During concentration polarization, the gel layer on the membrane surface superimposes its own rejection characteristics on those of the membrane. Usually, concentration polarization increases retention of lower-molecular weight species. A membrane with a 100K NMWL may reject 10 20% of albumin in a 0.1% solution of pure albumin. However, in the presence of larger solutes such as IgG, it may reject 90% of the albumin. Concentration polarization makes it very difficult to use UF for solute fractionation unless the solutes to be separated differ in size by at least an order of magnitude. Figure 1.4 Ultrafiltration separates proteins from soluble salts. Concentration polarization slows down filtration. The proteins form a gel layer on the membrane surface. 5

8 Flux (UF Flow Rate) During ultrafiltration, it is important to balance speed with retention to obtain optimal performance. A membrane s flux is defined as the flow rate divided by the membrane area. Using membranes with higher NMWL ratings will increase the flow, but at the same time lower the retention. A membrane should be selected for required rejection, consistent with desired flow rate. This is determined by surface area, macrosolute type, solubility, concentration and diffusivity, membrane type, temperature effects on viscosity and, to some extent, pressure. When concentration polarization is rate-controlling, flux is affected by solute concentration, fluid velocity, flow channel dimensions, and temperature. Effects of Operating Parameters on Flux Pressure When ultrafiltering dilute protein solutions or colloid suspensions, flux will increase with increasing transmembrane pressure (TMP). These effects are most apparent when operating under controlled positive pressure, such as when using a stirred cell. When the process is membrane-controlled (i.e., when the resistance of the gel layer is much smaller than that of the membrane), the flux-pressure relationship is linear. When the process is controlled by polarization (e.g., when the resistance of the gel layer is much larger than that of the membrane), flux will reach a plateau and may actually decrease with increases in pressure. Concentration When concentration of the retained species is very low, flux is independent of concentration. As solute concentration rises during operation, increased viscosity and the polarization effect cause flux to decrease. Temperature Increasing the operating temperature normally increases UF rates. A higher temperature increases solute diffusivity (typically 3 3.5% per degree Celsius for proteins) and decreases solution viscosity. Common practice is to operate at the highest temperature tolerated by the solutes and the equipment. An exception to the rule is fermentation broth concentration in the presence of some antifoams. Many antifoams exhibit a phenomenon called cloud point. As temperatures increase, antifoam comes out of solution, forming a second phase. Increasing temperature above the cloud point causes flux to decrease. ph Changing solution ph often changes molecular structure. This is especially true for proteins. At its isoelectric point, a protein begins to precipitate, causing a flux decrease. Fouling Flux decrease due to concentration polarization should not be confused with the effect of membrane fouling. Fouling is usually the deposition and accumulation of submicron particles and solute on the membrane surface and/or crystallization and precipitation of smaller solutes on or within the pores of the membrane. There may be a chemical interaction with the membrane. Importance of Recovery While rejection is used to characterize membrane performance, it does not always directly correlate with solute recovery from a sample or volume. Actual solute recovery the amount of material recovered after ultrafiltration is generally based on mass balance calculations. In many cases, especially when working with small samples of dilute, valuable solutions, the degree of recovery of a target solute is vitally important. In such cases, potential loss by nonspecific adsorption must be considered. Different membrane materials adsorb biomolecules to varying degrees. Where maximum recovery is desired, the choice of a membrane with the least non-specific adsorptivity is essential. Millipore s Ultracel regenerated cellulose membranes were specifically developed to minimize non-specific adsorption. Since adsorption is a direct function of membrane and device surface area, device size must be considered when recovery is important. Small, dilute samples should be concentrated with membranes of minimal surface area, commensurate with achievement of reasonable flow rates. Millipore offers a wide range of centrifugal devices, stirred cells, and tangential flow systems with an extensive choice of membrane areas and NMWLs. 6

9 Mode of Operation The pressure required for ultrafiltration can be supplied in a number of different ways depending on the product in use. For example, Millipore's small volume ultrafiltration products generally use centrifugal force. Pump pressure is used with the tangential-flow filtration (TFF) products and compressed gas is utilized with the stirred cell products. In addition, Millipore provides multiwell ultrafiltration products that utilize vacuum and centrifugation. Normal vs. Tangential Flow Filtration Filtration can be broken down into two different operational modes: normal flow filtration (NFF) and tangential flow filtration (TFF). The difference in fluid flow between these two modes is shown in Figure 1.5. Diafiltration Millipore membranes provide an inexpensive means of separating macromolecular mixtures into sizegraded classes either by direct ultrafiltration or by diafiltration. Diafiltration removes microsolutes by adding solvent to the solution being ultrafiltered at a rate equal to the UF rate, independent of microspecies concentration. This rapid, efficient process washes microspecies from the solution at constant volume, thereby purifying the retained species. This process is most effective if the passing molecules are at least 10 times smaller than the molecules to be retained and concentrated by the membrane. Diafiltration is useful for sample desalting and buffer exchange. When diafiltration is used for sample desalting or buffer exchange, there is no resulting change in buffer composition. A solution volume with 100 mm salt still contains 100 mm salt after the initial concentration spin. Rediluting the retentate with water and spinning again effectively decreases the salt concentration of the sample by the concentration factor of the ultrafiltration. For example, if a 4,000 µl sample containing 100 mm salt is concentrated to 50 µl (80X) in an Amicon Ultra centrifugal filter unit, rediluted with water to 4,000 µl, and reconcentrated, the salt concentration will be reduced 80X to 1.25 mm. To achieve more complete salt removal, multiple concentration and redilution spins are required. For most samples, two concentration/reconstitution cycles will remove about 99% of the initial salt content. With very small sample volumes, dilution of the sample before the initial concentration spin can often decrease salt concentration to an acceptable level. For example, if a 200 µl sample containing 100 mm salt is diluted to 4,000 µl before concentration in an Amicon Ultra centrifugal filter unit, the salt concentration in the 4,000 µl sample will be 5 mm. The concentrate will still contain 5 mm Figure 1.5 Normal flow filtration (NFF) vs. tangential flow filtration (TFF) In normal flow filtration (NFF), fluid is convected directly toward the membrane under an applied pressure. Particulates that are too large to pass through the pores of the membrane accumulate at the membrane surface or in the depth of the filtration media, while smaller molecules pass through to the downstream side. This type of process is Normal Flow Filtration Feed Flow Membrane Filtrate Pressure Tangential Flow Filtration Membrane Pressure Feed Flow Filtrate often called dead-end filtration. However, the term normal indicates that the fluid flow occurs in the direction normal to the membrane surface, so NFF is a more descriptive and preferred name. NFF can be used for sterile filtration of clean streams, clarifying prefiltration, and virus/protein separations. In tangential flow filtration (TFF), the fluid is pumped tangentially along the surface of the membrane. An applied pressure serves to force a portion of the fluid through the membrane to the filtrate side. As in NFF, particulates and macromolecules that are too large to pass through the membrane pores are retained on the upstream side. However, in this case the retained components do not build up at the surface of the membrane. Instead, they are swept along by the tangential flow. This feature of TFF makes it an ideal process for finer sized-based separations. Although TFF is more commonly associated with large scale processing, centrifugal UF devices with vertical membrane panels, such as Amicon Ultra devices, also benefit from a TFF-like mode of separation, particularly in a swinging bucket rotor. TFF is also commonly called cross-flow filtration. However, the term tangential is descriptive of the direction of fluid flow relative to the membrane, so it is the preferred name. 7

10 salt. If more complete salt removal is desired, a re-dilution/spin cycle should be added. In this example, if the original spin ended with 50 µl of retentate, redilution to 4,000 µl results in 0.06 mm salt concentration. The sample can then be reconcentrated to 50 µl in an Amicon Ultra centrifugal filter device. Diafiltration can be a continuous or a discontinuous process. In continuous diafiltration, such as in a stirred cell or a TFF device, the solution is maintained at a fixed volume while solvent flows continuously through the mixture. Salts and other microsolutes are steadily removed by convective transport. Microsolute exchange can be accomplished using the same principle. Constant operator attention is not required and the possibility of solute denaturation by overconcentration is eliminated. In discontinuous diafiltration, such as in a centrifugal ultrafiltration device, salts and microsolutes are removed by repeated concentration and dilution (Figure 1.6). Dialysis vs. Diafiltration Dialysis is a traditional method for removing microsolutes or exchanging solvents. It is a slow diffusive process generally employing regeneratedcellulose tubing as the barrier membrane. In dialysis, the process solution and exchange solvent are on opposite sides of the semi-permeable barrier membrane through which permeating microsolutes diffuse. The permeation rate of solutes from sample to dialysate is a direct ratio to the solute concentration and inversely proportional to the solutes molecular weight. Desalting by dialysis is timeconsuming and relatively inefficient at low concentrations. Millipore s Amicon centrifugal concentrators provide a fast, convenient, high-recovery alternative to dialysis or precipitation without diluting samples. The relative merits of diafiltration and dialysis are summarized in the Table 1.1. UF membrane 100 µl 10 µl Add 90 µl Milli-Q water 100 µl 10 µl 10X concentration of protein 90 µl 10-fold dilution 10X concentration of protein 90 µl 100 mm NaCl 100 mm NaCl 10 mm NaCl 10 mm NaCl Figure 1.6 Removing salts from retained solutes using diafiltration. The sodium chloride concentration is reduced by dilution. Table 1.1 Comparision of diafiltration and dialysis Diafiltration Transport convective with solvent, independent of microsolute composition. Rapid rate. Fractional removal independent of content. Ultrafiltration rate reduced with decreased temperature (net effect not as marked). At elevated macrosolute content, ultrafiltration rate reduced. Minimal exchange solvent required; easily contained in reservoir. Simple automation with endpoint control apparatus. Dialysis Transport diffusion-controlled, dependent on type of microsolute. Slow transport. Lower efficiency with decreased microsolute concentration. Marked temperature dependence (reduced transport at lower temperature). Microsolute transport relatively unaffected by macrosolute content. Frequent dialysate change. Recirculation about bags to maximize transport. Automation possible with complex equipment. 8

11 Guide to Selecting Membranes and Devices Millipore offers a complete range of centrifugal devices used for sample concentration, purification, and desalting or buffer exchange of soluble macromolecules. Millipore products are also available for applications such as cleaning up PCR reactions, separating protein-bound from free ligands, removing restriction enzymes, and recovering oligonucleotides from agarose gels. This section provides information to aid in choosing the correct product for a particular application. Membrane Selection Millipore offers three distinct types of membranes to choose from. This section will describe these three types of membranes and then provide information about choosing the correct membrane based on typical recoveries or application. Types of Membranes Ultracel Ultrafiltration Membrane To concentrate or desalt dilute solutions, use Ultracel series regenerated cellulose ultrafiltration membranes. The hydrophilic, tight microstructure of Ultracel membranes assures the highest possible retention with the lowest possible adsorption of protein, DNA or other macromolecules. Biomax Ultrafiltration Membrane To concentrate or desalt higher volumes of more concentrated samples (recommended for protein concentrations greater than 1.0 mg/ml), use Biomax polyethersulfone (PES) ultrafiltration membranes. Biomax membranes are recommended for samples such as serum, plasma, or conditioned tissue culture media. Durapore Microporous Membrane To clarify biological samples, recover DNA from agarose gels, retain chromatography resins or suspended solid media, use Durapore hydrophilic PVDF microporous membranes. Durapore membranes allow all soluble protein and nucleic acids to pass, retaining sub-cellular fragments, whole cell and particulate materials. Durapore membranes are extremely hydrophilic, and they provide the lowest binding of proteins and other biologicals of all commercially available microporous membranes. Membranes Organized by Typical Recoveries For globular proteins, there is a good correlation between molecular weight and Stoke s radius. This usually allows one to predict the recovery of a protein based on its molecular weight if a membrane with the same nominal molecular weight rating is used (see typical recovery of a panel of protein solutes in Table 2.1). However, in order to accommodate the wide range of potential protein solutes with different tertiary structures, we suggest initially using the rule of two to ensure optimal recovery. Rule of Two For Ultracel (regenerated cellulosic) membranes, Millipore recommends using a membrane with a NMWL at least two times smaller than the molecular weight of the protein solute that one intends to concentrate. Rule of Three For Biomax (polyethersulfone) membranes for stirred cells and TFF, Millipore recommends using Table 2.1 Membrane selection by recovery Cytochrome c BSA IgG NMWL (12.4 kda) (67 kda) (156 kda) 3K 10K 30K 50K 100K Recommended (>90% recovery) Typically >90% recovery, depends on solute Not recommended 9

12 a membrane with a NMWL at least three times smaller than the molecular weight of the protein solute that one intends to concentrate. Membranes Organized by Application See Table 2.2 to determine which centrifugal product to use based on application and membrane. Device Selection See Table 2.3 to determine which centrifugal product to use for protein concentration based on initial sample molecular weight and volume. Table 2.2 Membrane selection by application Ultrafiltration Membranes Type* Molecular Weight (NMWL) Microporous Membranes Pore Size (µm) Specialty Devices Protein concentration Protein purification/desalting/buffer exchange Desalting of column fractions Protein isolation from cell lysates Peptide concentration/desalting/buffer exchange Antibody concentration Virus concentration or removal Nucleic acid concentration/desalting/buffer exchange Oligonucleotide concentration/desalting/buffer exchange PCR cleanup Remove linkers prior to cloning Remove labeled nucleotides Antibody purification from hybridoma cells Rapid restriction mapping Clarify samples of particulate prior to HPLC Clarification of cell lysates and tissue homogenates Cell harvesting Natural product screening Restriction enzyme removal Bound vs. free drugs from serum/plasma (protein removal) DNA/RNA recovery from polyacrylamide gel DNA recovery from agarose gel Oligonucleotide recovery from polyacrylamide gel Removal of unincorporated label (e.g., fluorescein) from protein Removal of imidazole from His-tag fusion protein Ultracel Biomax 3K 5K 10K 30K 50K 100K Montage PCR Micropure-EZ Montage Gel Extraction *Selection of ultrafiltration membrane: Ultracel regenerated cellulose membrane is ideal for protein samples and nucleic acids. Biomax polysulfone membrane is ideal for complex samples (e.g., serum). 10

13 Table 2.3 Protein concentration devices by filtration capacity Filtration Capacity Millipore Device 0.5 ml 2 ml 4 ml 15 ml 20 ml 70 ml 1 L 2 L 10 L 10 L Small Volume Filtration Devices Microcon Centrifugal Filters* Ultrafree 0.5 Centrifugal Filters Ultrafree-MC Centrifugal Filters MultiScreen Filter Plate with Ultracel Membrane** Medium Volume Filtration Devices Centricon Centrifugal Filters Ultrafree-CL Centrifugal Filters Amicon Ultra-4 Centrifugal Filters Amicon Ultra-15 Centrifugal Filters Centriprep Centrifugal Filters Centriplus Centrifugal Filters Centricon Plus-20 Centrifugal Filters Large Volume Filtration Devices Centricon Plus-70 Centrifugal Filters Amicon Stirred Cells Series 8000, High Output, Solvent-Resistant Stirred Cells Tangential Flow Filtration (TFF) Pellicon XL 50 Ultrafiltration Devices Prep/Scale Spiral Wound UF Modules Pellicon-2 Ultrafiltration Modules *Bold type indicates recommended devices. **96-well plate. Volume per well. 11

14 Small Volume Devices Ultrafiltration Devices Microcon Centrifugal Filters Maximum initial volume: 500 µl Typical final volume: 5 µl NMWLs: 3K, 10K, 30K, 50K, 100K Microcon centrifugal filter devices, the lab standard, simply and efficiently concentrate and desalt macromolecular solutions using any centrifuge that can accept 1.5 ml tubes. The device s lowadsorption components and its Ultracel-YM membrane, together with the patented invert recovery spin, combine to yield unusually high recovery rates typically > 95%, with concentration factors as high as 100X. The invert spin also permits the recovery of low volumes. Ultrafree-0.5 Centrifugal Filters Maximum initial volume: 500 µl Typical final volume: 50 µl NMWLs: 5K, 10K, 30K, 50K, 100K Ultrafree-0.5 centrifugal filter devices process aqueous biological solutions using any centrifuge that accommodates 2.2 ml centrifuge tubes. The device s vertical Biomax PES membrane configuration, parallel to the direction of the centrifugal force, reduces concentration polarization. This allows for the highest possible flow rates, even in solutions with high levels of particles. Concentrated protein is retrieved by pipette from a concentrate pocket located below the membrane surface. MultiScreen Filter Plates with Ultracel-10 Membrane Maximum initial volume: 500 µl per well NMWL: 10K MultiScreen Filter Plates with Ultracel-10 Membrane are the first automationcompatible, high throughput ultrafiltration plates for protein purification. The 10K NMWL Ultracel regenerated cellulose ultrafiltration membrane provides low non-specific binding and high protein recovery (>95% retention of Cytochrome c). Microfiltration Devices Ultrafree-MC Centrifugal Filters Maximum initial volume: 400 µl Typical final volume: 5 µl Pore sizes: 0.1 µm, 0.22 µm, 0.45 µm, 0.65 µm, 5.0 µm Ultrafree-MC centrifugal filter units are disposable devices used for sample clarification with high recovery of biological solutions in the 0.05 to 0.5 ml range. Ultrafree-MC units are available in a range of microporous and ultrafiltration membranes and are for single use only. They fit standard microcentrifuge, fixed-angle rotors capable of holding 1.5 ml or 20 mm outer diameter (O.D.) tubes. Ultrafree-MC centrifugal filter units provide fast filtration and highly reproducible performance. 12

15 Medium Volume Devices Ultrafiltration Devices Amicon Ultra-4 Centrifugal Filters* Maximum initial volume: 4 ml Typical final volume: 50 µl NMWLs: 5K, 10K, 30K, 50K, 100K Amicon Ultra centrifugal filters are the premier tool for protein concentration, desalting, and removing macromolecules and precipitates. The devices combine Ultracel low-binding ultrafiltration membrane with a vertical housing for fast sample processing and typical sample recoveries of >90%. For volumes up to 4 ml, the Amicon Ultra-4 device provides fast ultrafiltration with the capability for high concentration factors. Centricon Centrifugal Filters Maximum initial volume: 2 ml Typical final volume: 25 µl NMWLs: 3K, 10K, 30K, 50K, 100K Centricon centrifugal filter devices provide fast, efficient concentration and desalting of macromolecular solutions by ultrafiltration through low-adsorption, hydrophilic Ultracel-YM regenerated cellulose membranes. Designed for use in centrifuges with fixed-angle rotors, they can provide up to 80-fold sample enrichment with minimal solute loss by adsorption. Centriprep Centrifugal Filters Maximum initial volume: 15 ml Typical final volume: 700 µl NMWLs: 3K, 10K, 30K, 50K Centriprep centrifugal filter devices are disposable ultrafiltration devices used for concentrating and desalting high solute biological samples. These complete, ready-to-use ultrafiltration devices are designed for operation in most centrifuges that can accommodate 50 ml centrifuge tubes. Centriprep devices are best suited for applications in which concentration factors are so high that they might affect protein precipitation or aggregation. Centriplus Centrifugal Filters Maximum initial volume: 15 ml Typical final volume: 500 µl NMWLs: 3K, 10K, 30K, 50K, 100K The disposable Centriplus centrifugal filter device is used for the concentration and desalting of macromolecular solutions with up to 100-fold sample enrichment with minimal loss of solute. These devices are designed for use in most centrifuges that can accommodate 50 ml centrifuge tubes, with swingingbucket or fixed-angle rotors. Amicon Ultra-15 Centrifugal Filters* Maximum initial volume: up to 15 ml Typical final volume: 200 µl NMWLs: 5K, 10K, 30K, 50K, 100K Amicon Ultra-15 centrifugal filters contain a high recovery Ultracel membrane and are used to concentrate biological samples, to purify macromolecular components found in tissue culture extracts or cell lysates, and for desalting and buffer exchange. The devices can concentrate and desalt 15 ml in as few as 10 minutes. Centricon Plus-20 Centrifugal Filters Maximum initial volume: 20 ml Typical final volume: 200 µl NMWLs: 5K, 10K, 30K, 100K Centricon Plus-20 centrifugal filter units are disposable, single-use devices designed for rapid processing of aqueous biological solutions. The devices can concentrate most 20 ml solutions down to a minimum volume of 200 µl. Microfiltration Devices Ultrafree-CL Centrifugal Filters Maximum initial volume: 2 ml Typical final volume: 10 µl Pore sizes: 0.1 µm, 0.22 µm, 0.45 µm, 0.65 µm, 5.0 µm Ultrafree-CL centrifugal filter units are disposable, single-use devices used to process aqueous biological solutions. Ultrafree-CL units can be used in standard, variablespeed centrifuges with fixed-angle rotors capable of holding 15 ml tubes. A complete device consists of a filter cup and a filtrate collection tube with cap. Ultrafree-CL units are available in a range of microporous and ultrafiltration membranes. *Amicon Ultra-4 and 15 are the first centrifugal devices to combine high speed and high recovery. They are highly recommended for most protein concentration and desalting applications. 13

16 Large Volume and TFF Devices Ultrafiltration Devices Centricon Plus-70 Centrifugal Filters Maximum initial volume: 70 ml Typical final volume: 350 µl NMWLs: 5K, 10K, 30K, 100K Centricon Plus-70 centrifugal filter units concentrate most 70 ml solutions down to 350 µl in just 25 minutes, making them a convenient alternative to dialysis, lyophilization, precipitation techniques, or stirred cells. Samples are typically concentrated in the 50X to 200X range, depending on the sample type and starting sample volume. Tangential Flow Filtration (TFF) Pellicon XL Devices Maximum initial volume: 2 L NMWLs: 5K, 8K, 10K, 30K, 50K, 100K, 300K, 500K, 1000K Pellicon XL devices are small volume tangential flow filtration (TFF) devices offering performance, ease of use, and linear scalability for pharmaceutical and biotechnology development applications. Pellicon XL devices are available in a wide selection of membrane types and cut-offs. An optional graduated 100 ml reservoir is available for simple small-volume separations. Prep/Scale Spiral Wound Ultrafiltration Modules Maximum initial volume: 2 L to 10 L NMWLs: 1K, 3K, 5K, 10K, 30K, 50K, 100K, 300K Prep/Scale modules are used in conjunction with a peristaltic pump, flexible tubing and feed solution in order to economically prepare laboratory scale samples up to small production volumes by tangential flow filtration. Highly reliable and consistent, Prep/Scale modules offer cassette-like efficiencies with reproducible separations performance. Pellicon 2 Modules Volumes >10 L NMWLs: 1K, 3K, 5K, 8K, 10K, 30K, 50K, 100K, 300K, 500K, 1000K Pellicon 2 modules offer true linear scalability from laboratory size cartridges to industrial cartridge assemblies for processing thousands of liters. Pellicon 2 modules facilitate predicatable and faster scale-up with less effort and are available with membranes to match virtually any separation challenge. Stirred Cells Maximum initial volume: 2,000 ml Typical final volume: 3 ml NMWLs: 1K, 3K, 10K, 30K, 50K, 100K, 300K, 500K Stirred Cells are ideal for concentration, diafiltration and buffer exchange of macromolecule solutions including proteins, enzymes, antibodies and viruses. They are capable of rapid concentration or salt removal followed by concentration in the same unit. Millipore offers three types of stirred cells: Series 8000 Stirred Cells Five different sizes handle volumes from 3 ml to 400 ml High flow rates with solutions up to 10% macrosolute concentration High-Output Stirred Cells For 150 mm disc filters Large membrane area Solvent-Resistant Stirred Cells For 47 and 76 mm disc filters Borosilicate glass cylinder and PTFE components for broad compatibility Series 8000, High-Output and Solvent-Resistant Stirred Cells 14

17 Specialty Devices and Kits Montage DNA Gel Extraction Kit and Ultrafree-DA Centrifugal Filters Designed to extract DNA fragments that are 100 to 10,000 bp in size; 0.45 µm Durapore membrane The Montage DNA Gel Extraction Kit is a fast, effective solution for fully functional DNA recovery from agarose gel slices. In one 10-minute spin, the agarose gel material containing the DNA of interest is fragmented and compressed to extrude the DNA that is ready for sequencing or cloning. The Montage Gel Extraction kit contains all the necessary consumables and the kit protocol is optimized for use with standard agarose gels (<1.25% concentration). Ultrafree-DA centrifugal devices available separately. Montage PCR Centrifugal Filters Maximum initial volume: 500 µl Typical final volume: 100 µl NMWLs: 3K, 10K, 30K, 50K, 100K The Montage PCR filter unit is a convenient method for single-sample PCR purification. The high-performance device purifies PCR products from salts and primers in a single centrifugation step. Purified samples are ready for downstream applications with no additional purification steps. Micropure -EZ Centrifugal Filters Maximum initial volume: 250 µl Holdup volume: < 5 µl NMWLs: 3K, 10K, 30K, 50K, 100K Micropure-EZ centrifugal filters provide an easy, rapid means of removing restriction and other enzymes from double-stranded (ds) DNA. In a single 30-second spin, enzymes from the reaction mix are selectively adsorbed by the membrane and DNA is recovered in a vial undiluted and enzyme-free. If concentration or desalting of the sample is required, a Microcon centrifugal filter device may be substituted for the filtrate vial, providing for DNA purification, concentration and desalting in a single unit. DNA recovery is not affected by the binding capacity of an adsorption matrix. Micropure-EZ devices will deliver a minimum of 85% recovery of dsdna; higher recoveries can be achieved by adding a rinsing step. Centrilutor Micro-Electroeluters Maximum initial volume: 2 ml Typical final volume: 25 µl NMWLs: 3K, 10K, 30K, 50K, 100K The Centrilutor Micro-Electroeluter elutes proteins from gel slices and then removes the buffer and concentrates the sample using Centricon centrifugal filter devices. This fast, easy way to recover 1 25 µg of protein from gel slices requires no additional handling of the sample once the gel slices are placed into the eluter. 15

18 Protocols Proteins Concentration, Desalting, and Buffer Exchange with Amicon Ultra or Microcon Centrifugal Filters Introduction Amicon centrifugal devices from Millipore are ideal for removal or exchange of salts, sugars, nucleotides, and non-aqueous solvents, as well as other materials of low molecular weight. They also serve to separate free from bound species. Millipore centrifugal concentrators provide fast, convenient, high-recovery alternatives to dialysis and ethanol precipitation. Sample dilution, often associated with spin columns, is not a problem. Salt transfer across the membrane is efficient and independent of microsolute concentration or size. Millipore s Amicon Ultra-4 and -15 centrifugal filters are designed for high speed with high recovery. The devices incorporate low-binding Ultracel regenerated cellulose ultrafiltration membrane for sample concentration and purification of solutions containing dilute or purified protein solutes, antigens, antibodies, enzymes, or microorganisms. Their speed and excellent recovery make them ideal for desalting and buffer exchange applications. One of the most common applications for Amicon Ultra devices is concentration and desalting of column fractions during protein purification by various chromatography methods. Two examples below demonstrate the use of Amicon Ultra devices for high protein and enzymatic activity recovery. Method 1. Select the device with the appropriate NMWL and volume for the application. 2. Add the sample to the reservoir of the centrifugal device. 3. If the sample is smaller than the maximum volume, it can be diluted up to the maximum volume before the first centrifugation step. This will help increase the salt removal. 4. Centrifuge at the specified g-force for the recommended amount of time. 5. Remove the initial filtrate from the filtrate tube and set aside. 6. Add enough buffer or water to the device to bring the sample volume up to 4 or 15 ml. 7. Centrifuge again. 8. Set aside the filtrate. 9. Recover the concentrated, de-salted sample. NOTE: Both of the filtrates should be retained until the concentrated sample has been analyzed. Table 3.1 Removal of sodium chloride and recovery of protein with Amicon Ultra-15 device Cytochrome c Cytochrome c BSA BSA IgG 0.25 mg/ml 0.25 mg/ml 1 mg /ml 1 mg/ml 1 mg/ml NMWL 5 kda 10 kda 30 kda 50 kda 100 kda Spin % Protein % NaCl % Protein % NaCl % Protein % NaCl % Protein %NaCl % Protein % NaCl Recovery Removal Recovery Removal Recovery Removal Recovery Removal Recovery Removal Three Amicon Ultra-15 devices of each cut-off were tested with 15 ml of solute. 500 mm NaCl was added to each solution. Each spin was performed at 4000 x g for 30 minutes. After the first spin, the retentate was brought up to 15 ml with ultrapure water from a Milli-Q (Millipore) system. OD readings were taken at 410 nm for Cytochrome c and 280 nm for BSA and IgG. 16

19 Results The transfer of salts across a membrane filter is independent of sample concentration or size. There is no change in the composition of the buffer when desalting using ultrafiltration. For example, a solution containing 500 mm salt still contains that concentration after the initial centrifugation. Adding another volume of salt-free buffer or water to the retentate and centrifuging again will reduce the salt concentration. This process, known as diafiltration, can be repeated to achieve maximum salt removal. Diafiltration can also be used when it is desirable to have the sample in a different buffer. The sample is concentrated and then repeatedly diluted with the desired buffer and concentrated again. As the results show in Tables , the efficient design of the Millipore devices allowed >90% of the salt to be removed during the first centrifugation step. Typically, only one subsequent centrifugation step was needed to increase the typical salt removal to 99% with >90% recovery of the sample. Protein purification by chromatography usually involves the collection of multiple column fractions, with only some of those fractions containing the protein of interest. After the fractions are combined, a protein concentration step is often required for protein storage, or concentration with buffer exchange may be needed for downstream separations. Table 3.2 Removal of sodium chloride and recovery of protein with Amicon Ultra-4 device Cytochrome c Cytochrome c BSA BSA IgG 0.25 mg/ml 0.25 mg/ml 1 mg /ml 1 mg/ml 1 mg/ml NMWL 5 kda 10 kda 30 kda 50 kda 100 kda Spin % Protein % NaCl % Protein % NaCl % Protein % NaCl % Protein %NaCl % Protein % NaCl Recovery Removal Recovery Removal Recovery Removal Recovery Removal Recovery Removal Three Amicon Ultra-4 devices of each cut-off were tested with 4 ml of solute. 500 mm NaCl was added to each solution. Each spin was performed at 4000 x g for 10 minutes. After the first spin, the retentate was brought up to 4 ml with ultrapure water from a Milli-Q (Millipore) system. OD readings were taken at 480 nm for Cytochrome c and 280 nm for BSA and IgG. Table 3.3 Removal of sodium chloride and recovery of IgG with Centricon Plus-20 filter device with Ultracel-PL 30 membrane Spin % NaCl % IgG Number Remaining Recovered ml of 1 mg/ml bovine IgG in 500 mm NaCl was concentrated to 150 µl. Sample was spun twice at 2000 x g in a swinging bucket rotor at 25 C for 20 minutes. Table 3.4 Removal of riboflavin and recovery of IgG with Microcon filter device with Ultracel-YM membrane Spin % Riboflavin % IgG Number Remaining Recovered < < µl of a 50:50 mixture of riboflavin and IgG were spun in a Microcon 3K NMWL device at 12,000 x g for 75 minutes at room temperature in 55 angle rotor. After the initial spin, the retentate was twice diluted with 500 µl of PBS and spun again. After each spin, concentration of riboflavin and IgG in the filtrate and retentate were monitored. 17

20 Concentration of Indoleamine 2,3-Dioxygenase Courtesy of Eduardo Vottero, University of British Columbia Indoleamine 2,3-dioxygenase (IDO; MW 48,000) is a heme-containing enzyme that is the first and rate-limiting enzyme in human tryptophan metabolism. IDO processes 98% of the total tryptophan available in the human body and is critical in suppression of immunoresponse by blocking T-lymphocyte proliferation locally [Swanson et al, Am J Respir Cell Mol Biol [manuscript in preparation] (2003); Sarkhosh et al, J Cell Biochem 90, 206 (2003); Mellor et al, J Immunol 171, (2003)]. Recombinant IDO was expressed in E. coli BL21 (DE3) cells utilizing the pet 28a (+) vector system. In this system, a hexahistidyl tag was fused to fullsize IDO at the N-terminus with a spacer sequence and a thrombin cleavage site. The protein was purified by conventional His-tag purification methods and eluted with imidazole. The histidine tag was removed by thrombin cleavage. Final purification was done by gel filtration chromatography G-75. Amicon Ultra-15 centrifugal devices were used to 40 kda 35 kda Figure 3.1 SDS-PAGE of purified indoleamine 2,3-dioxygenase before and after concentration using Amicon Ultra-15 centrifugal devices. concentrate the IDO fractions from an initial concentration of ~0.5 mg/ml to a final concentration of 10 mg/ml. Samples were analyzed by SDS-PAGE using a 12.5% polyacrylamide gel (Figure 3.1). In addition, it was shown that no IDO activity loss was observed after concentration using an Amicon Ultra device. Concentration of PKR and Buffer Exchange Courtesy of Peter A. Lemaire and Dr. James Cole, University of Connecticut Human protein kinase R (PKR) is one of the major proteins induced by interferon as part of the host defense against viral infection 1 4. PKR is synthesized in a latent form and is activated by autophosphorylation induced upon binding dsrna. Once phosphorylated, active PKR phosphorylates the eukaryotic translation initiation factor elf2α leading to a block in protein synthesis in virally infected cells. PKR has been implicated as a participant in various signal transduction pathways associated with cellular processes including transcription 7 9, differentiation 10, apoptosis 11, splicing 14 and transformation 5,6. However, difficulties in purifying PKR in large amounts has limited rigorous biophysical characterization of the mechanisms of PKR activation. A high-yield prokaryotic expression system has been developed for PKR, and PKR has been purified using three chromatography steps on Agarose-Heparin, Agarose-Poly (I), Poly (C) and Sephacryl S-200 gel filtration columns. After the last step, PKR-containing column fractions were pooled and concentrated using Amicon Ultra-15 30K NMWL devices. The concentration step was necessary for long-term protein storage. Table 3.5 shows the protein recovery results obtained after four concentrations. Over 90% recovery was obtained and no protein loss to the filtrate was observed. Table 3.5 PKR concentration results Spin 1 Spin 2 Spin 3 Spin 4 Starting volume (ml) Starting concentration (mg/ml) Volume of retentate (µl) Total amount of PKR (mg) Before UF After UF Filtrate % Recovery

21 Amicon Ultra-15 30K NMWL devices were also used for exchanging buffer for PKR autophosphorylation (activation) assay. 200 µl of mg/ml PKR in Protein Storage buffer (20 mm HEPES, 1 M NaCl, 10 mm β-mercaptoethanol, 0.1 mm EDTA, 10% glycerol, ph 7.5) was diluted to 15 ml with Phosphorylation Buffer (20 mm HEPES, 50 mm KCl, 5 mm MgCl 2, 0.1 mm EDTA, 1 mm DTT, ph 7.5) and re-concentrated three times using Amicon Ultra devices at 3000 x g for 20 minutes at 4 C. The filtrates from the three steps were pooled and the total amount of protein in all samples was determined by UV absorption A 280. Protein activity was tested by autophosphorylation assay. The protein in the storage buffer was supplemented with 5 mm MgCl 2 and the samples were allowed to undergo autophosphorylation at 30 C for 20 minutes in the presence of 3 mm ATP and 3 µci [γ 32 P] ATP. The activity was determined by autoradiography and quantified by liquid scintillation counting. As shown in Figure 3.2, the activity of PKR when no buffer exchange was only 6% of that when buffer exchange step was performed. Hence, the UF successfully exchanged the buffer while maintaining the activity of the protein. References 1. Samuel CE. Antiviral actions of interferon. Interferon-regulated cellular proteins and their surprisingly selective antiviral activities. Virology 1991;183(1): Hovanessian AG. The double stranded RNAactivated protein kinase induced by interferon: dsrna-pk. J Interferon Res 1989;9(6): Relative Activity of PKR No Buffer Exchange Buffer Exchange 3. Lebleu B, et al. Interferon, double-stranded RNA, and protein phosphorylation. Proc Natl Acad Sci USA 1976;73(9): Samuel CE. Mechanism of interferon action: phosphorylation of protein synthesis initiation factor eif-2 in interferon-treated human cells by a ribosome-associated kinase processing site specificity similar to hemin-regulated rabbit reticulocyte kinase. Proc Natl Acad Sci USA 1979;76(2): Koromilas AE, et al. Malignant transformation by a mutant of the IFN-inducible dsrna-dependent protein kinase. Science 1992;257: Meurs E, et al. Tumor supressor function of interferon-induced double-stranded RNA activated protein kinase. Proc Natl Acad Sci USA 1993;90: Wong AH, et al. Physical association between STAT1 and the interferon-inducible protein kinase PKR and implications for interferon and doublestranded RNA signaling pathways. EMBO J, 1997;16(6): Cuddihy AR, et al. Double-stranded-RNAactivated protein kinase PKR enhances transcriptional activation by tumor suppressor p53. Mol Cell Biol 1999;19(4): Demarchi F, Gutierrez MI, Giacca M. Human immunodeficiency virus type 1 Tat protein activates transcription factor NF-kappaB through the cellular interferon-inducible, double-stranded RNA-dependent protein kinase, PKR. J Virol 1999; 73(8): Petryshyn R, et al. Effect of interferon on protein translation during growth stages of 3T3 cells. Arch Biochem Biophys 1996;326(2): Barber GN. Host defense, viruses and apoptosis. Cell Death Differ 2001;8(2): Tan SL, Katze MG. The emerging role of the interferon-induced PKR protein kinase as a apoptotic effector: A new face of death? J Interferon Cytokine Res 1999;19: Balachandran S, et al. Activation of the dsrnadependent protein kinase, PKR, induces apoptosis through FADD-mediated death signaling. EMBO J, 1998;17(23): Osman F, et al. A cis-acting element in the 3'-untranslated region of human TNF-alpha mrna renders splicing dependent on the activation of protein kinase PKR. Genes Dev 1999;13(24): Figure 3.2 Comparison of PKR autophosphorylation with buffer exchange by ultrafiltration and without buffer exchange. 19

22 Protocols Proteins Detergent Removal with Microcon, Amicon Ultra or Centricon Centrifugal Filters Introduction Microcon, Amicon Ultra and Centricon centrifugal filters are efficient laboratory tools for removing small molecules from solutions of proteins or nucleic acids. Often, the molecule to be removed is one of a number of commonly used detergents or protein solubilizing agents. The chemical nature of most detergents allows for micelle formation above a critical concentration limit (Critical Micelle Concentration, CMC). Micelle formation results in aggregation of the detergent and leads to gross changes in molecular structure. This affects the amount of the detergent that can be removed from a solution by centrifugal devices with specific nominal molecular weight limit (NMWL) membranes. For example, the monomer of Triton X-100 has a molecular weight of daltons. Triton X-100 should pass readily through the 10,000 NMWL membrane in an Amicon Ultra device. However, at concentrations above 0.01% (0.2 mm), Triton X-100 forms micelles composed of approximately 140 monomeric units. During ultrafiltration, the micelles behave like 70,000 90,000 dalton globular proteins. As a result, more than 90% of Triton is retained by the ultra-filtration membrane. Therefore, above the CMC of Triton X-100, an Amicon Ultra-4 100K NMWL concentrator would be required to remove the detergent effectively. Method and Results In a series of studies, Millipore researchers used Total Organic Carbon (TOC) analysis to measure detergent removal by Microcon, Amicon Ultra and Centricon concentrators after a single centrifugation spin (note that complete detergent removal generally requires 3 5 spins). As the results in the Tables 4.1 and 4.2 indicate, detergent removal depends both on the original detergent concentration and the NMWL of the centrifugal units. All measurements shown were made using detergent/distilled water solutions. NOTE: Temperature, the presence of salts in the solution, and/or macromolecule/detergent interactions may lower the CMC for a particular detergent. Use the tables only as general guidelines in assessing the efficiency of detergent removal with the Microcon, Amicon Ultra and Centricon devices. 20

23 Table 4.1 Percent detergent removal after one spin with Centricon and Microcon centrifugal devices NMWL Detergent 3kDa 10 kda 30 kda 50 kda 100 kda SDS 0.01% > 90% > 90% > 90% > 90% > 90% 0.1% > 90% > 90% > 90% > 90% > 90% 1% 40 89% 40 89% 40 89% 40 89% 40 89% 5% < 40% < 40% < 40% 40 89% 40 89% NaDeoxycholate 0.1% > 90% > 90% > 90% > 90% > 90% 1% > 90% > 90% > 90% > 90% > 90% 5% 40 89% 40 89% 40 89% > 90% > 90% CAPS 5% > 90% > 90% > 90% > 90% > 90% CPCl % > 90% > 90% > 90% > 90% > 90% 0.1% 40 89% 40 89% 40 89% 40 89% 40 89% 1% < 40% < 40% < 40% < 40% 40 89% 5% < 40% < 40% < 40% < 40% 40 89% TDMABr 2 0.1% > 90% > 90% > 90% > 90% > 90% 1% < 40% < 40% < 40% 40 89% > 90% 5% < 40% < 40% < 40% < 40% > 90% Digitonin 0.01% > 90% > 90% > 90% > 90% > 90% 0.1% 40 89% 40 89% 40 89% 40 89% 40 89% 1% < 40% < 40% < 40% < 40% < 40% Tween % < 40% < 40% 40 89% 40 89% > 90% 0.1% < 40% < 40% < 40% 40 89% > 90% 1% < 40% < 40% < 40% < 40% 40 89% 5% < 40% < 40% < 40% < 40% < 40% Triton X % 40 89% 40 89% 40 89% 40 89% > 90% 0.10% < 40% < 40% < 40% < 40% > 90% 1% < 40% < 40% < 40% < 40% 40 89% 5% < 40% < 40% < 40% < 40% < 40% CHAPS 0.10% > 90% > 90% > 90% > 90% > 90% 1% 40 89% 40 89% > 90% > 90% > 90% 5% < 40% < 40% 40 89% > 90% > 90% Table 4.2 Percent detergent removal after one spin with Amicon Ultra-4 centrifugal devices NMWL Detergent 10 kda 30 kda SDS 0.1% 95% 98% 1% 38% 48% 5% 94% 95% Tween % 30% 42% 1% 28% 35% 5% 82% 77% Triton X % 3% 47% 1% 2% 20% 5% 2% 20% CHAPS 0.1% 90% 96% 1% 66% 93% 5% 38% 73% 1 Cetylpyridinium chloride 2 Tetradecyltrimethylammonium bromide 21

24 Protocols Proteins Two-Dimensional Electrophoresis Sample Preparation with Amicon Ultra Centrifugal Filters Two-dimensional electrophoresis (2DE) is one of the most commonly used methods in proteome analysis. Briefly, proteins are separated by their isoelectric point (first dimension separation) followed by SDS- PAGE separation by molecular weight (second dimension separation). Although it is a powerful method for simultaneously displaying hundreds of proteins, 2DE presents a challenge for sample preparation. Salts and ionic detergents are common chemicals contaminating biological samples that are not compatible with 2DE separation; however they are often required to solubilize proteins from cells and tissues. The high concentration of salts combined with the relatively low protein content make samples completely unsuitable for isoelectric focusing. Usually protein concentration is achieved by protein precipitation with acetone or TCA. The disadvantage of protein precipitation is that some of the proteins become insoluble and can not be resolubilized in IPG buffer. Another disadvantage is that many salts become insoluble in acetone and precipitate along with the proteins. Ultrafiltration (UF) can achieve protein concentration and desalting in one step. Figures 5.1 and 5.2 present two examples of protein preparation for 2DE by acetone precipitation and ultrafiltration. Both examples demonstrate that UF provides more efficient salt removal and allows better separations and improved resolution of protein spots in two-dimensional electrophoresis. Figure 5.1 Two-dimensional electrophoresis of yeast cell lysate prepared by acetone precipitation (left) and ultrafiltration (right). Sacharomyces cerevisiae strain s288c was grown to log phase. The cells were pelleted and resuspended in Cellular and Organelle Membrane Solubilizing Reagent from the ProteoPrep kit (Sigma) and lysed by sonication. Cellular debris was removed by centrifugation and the supernatants were reduced and alkylated with 5 mm tributylphosphine and 10 mm acrylamide. Lysates were acetone-precipitated to remove residual Tris and alkylating reagent or were filtered through Amicon Ultra-4 10K NMWL devices. Samples were redissolved in ProteomIQ Resuspension Reagent (Proteome Systems), focused in broad range (ph 3 10) immobilized ph gradients (IPGs) on the IsoelectrIQ IEF instrument. Second dimension gels were 6 15% polyacrylamide gradient GelChips (Proteome Systems) run on an ElectrophoretIQ 2D instrument (Proteome Systems). Data courtesy of Dr. G. Smejkal, Proteome Systems, Inc., Woburn, MA, USA. 22

25 Figure 5.2 Two-dimensional electrophoresis of endothelial cell lysates prepared by acetone precipitation (left) and ultrafiltration with Amicon Ultra devices (right). Whole cell lysates of endothelial cells were prepared in 7 M urea, 2 M thiourea, 4% CHAPS, 10 mm DTT, 20 mm Tris buffer. The samples were very dilute and presumably contained DNA fragments, salts and lipids. 300 mg of the total protein was either acetone-precipitated or desalted in Amicon Ultra devices. After acetone precipitation with four volumes of cold acetone, samples were incubated overnight at 20 C, precipitated by centrifugation, rinsed with cold acetone, and pelleted again. The resulting pellet was resuspended in rehydration buffer (7 M urea, 2 M thiourea, 4% CHAPS, 10 mm DTT, 20 mm Tris). Alternatively, samples were filtered through Amicon Ultra-4 10K NMWL devices after being mixed with nine volumes of 20 mm Tris HCl, ph 7. The sample was concentrated to approximately 40 µl and adjusted to 350 µl with rehydration buffer. The proteins were separated by IEF in 18 cm IPG Drystrips, ph 3 10 (Amersham). A second dimension separation was performed in 10% SDS-PAGE gel. The gels were silver stained and scanned. Data courtesy of Dr. Leonid Kryazhev, Genome Quebec, Montreal, Canada. 23

26 Protocols Proteins Purification of Serum Peptides for Biomarker Research with Amicon Ultra Centrifugal Filters Introduction A biomarker can be defined as a molecule that indicates an alteration in physiology. Biomarkers play an essential role in the drug discovery and development process. They provide powerful clues to genetic susceptibility, disease progression, and predisposition, as well as drug response and the physiological and metabolic profiles of diseases and drug responses. Biomarkers can also provide valuable diagnostic and prognostic information that can facilitate personalized medicine. Peptide and protein patterns have been linked to ovarian cancer, breast cancer, prostate cancer and astrocytoma 1 5. Most diagnostic tests are based on blood or urine analysis 5. Serum is a key source of putative protein biomarkers, and, by its nature, can elucidate organ-confined events. Use of mass spectrometry coupled with bioinformatics has been demonstrated as being capable of distinguishing between serum protein pattern signatures in late-stage and earlystage ovarian cancer patients 6. One of the major impediments to the discovery of new biomarkers is the presence of salts, proteins, and lipids in plasma or serum that makes it difficult to detect and analyze peptides by mass spectrometry. When untreated serum is spotted onto a MALDI-TOF plate, it does not produce any useable signal in mass spectrometry. Multiple protocols have been developed to extract and enrich peptides from tissues and body fluids, such as batch reversed phase chromatography over C18 resin and extraction with 0.1% trifluoroacetic acid (TFA) or 50% acetonitrile to selectively precipitate large proteins while enhancing the solubility of smaller proteins and peptides. Ultrafiltration has previously been reported as a sample preparation tool to prepare low molecular weight fractions for biomarker analysis In this study we show that ultrafiltration in combination with solid phase extraction (SPE) on C18 resin can be a convenient and efficient method for serum peptide purification. The approach provides more peptides for mass spectrometry analysis than the acetonitrile precipitation method. Methods Preparation of Serum Peptides by UF and SPE One milliliter of human serum, with or without acetonitrile, was filtered using Amicon Ultra-4 10K NMWL centrifugal devices. The devices were centrifuged in a swinging bucket rotor for 15 to 30 minutes at 3000 x g. Ten microliters of the filtrate was acidified with 5 µl of 1% TFA, concentrated with ZipTip µc18 pipette tips. Co-elution was performed directly onto a MALDI target with 2 µl of -cyano-4-hydroxycinnamic acid matrix (5 mg/ml in 50% acetonitrile, 0.1% TFA). If acetonitrile was added to the serum prior to the filtration, the samples were briefly evaporated in a Speed-Vac centrifuge to remove solvent before ZipTip purification. Peptide Analysis by Mass Spectrometry Peptide-containing ultrafiltrates from cell lysates or human serum were acidified with 1% TFA and concentrated on ZipTip µc18 or ZipTip SCX pipette tips following the procedure outlined in the user guide. All samples were overlaid with 1 µl of -cyano-4-hydroxycinnamic acid matrix (5 mg/ml in 50% acetonitrile, 0.1% TFA) and analyzed on Voyager-DE Workstation (Applied Biosystems) in linear mode. Preparation of Serum Peptides by Acetonitrile Precipitation Acetonitrile was added to human serum at a 1:1 ratio (v:v), and samples were centrifuged to precipitate larger proteins. The supernatant was dried in a Speed-Vac centrifuge, resuspended in 0.1% TFA, and then desalted and concentrated with ZipTip µc18 pipette tips. Co-elution was performed directly onto a MALDI target with 2 µl of -cyano-4-hydroxycinnamic acid matrix (5 mg/ml in 50% acetonitrile, 0.1% TFA). 24

27 Results While human serum contains numerous peptides and small proteins, they are not accessible by direct mass spectrometry analysis. Even after reverse phase concentration and desalting, only a few peptides are detectable in the mass spectrum (data not shown). This can be explained by the high concentration of proteins and lipids competing with the peptides to bind to the resin (Figure 6.1A). One of the common methods for serum peptide preparation for mass spectrometry is acetonitrile fractionation, where the addition of 50 70% acetonitrile precipitates larger proteins, while the peptides stay soluble in the supernatant. Another way to produce relatively protein-free filtrates is ultrafiltration. Figure 6.1 presents the MALDI-TOF spectra of (A) straight rat serum; (B) serum supernatant after 50% acetonitrile precipitation; and (C) serum ultrafiltrate processed with an Amicon Ultra-4 30K NMWL device. We observed higher quality spectra and an increased number of MALDI- TOF detected peptides if the serum was ultrafiltered. Further improvement of spectra can be achieved by reverse phase chromatography, which concentrates and desalts the peptides. The ZipTip µc18 pipette tip is a convenient and efficient tool for micro-scale sample preparation prior to mass spectrometry. Figure 6.2 shows the MALDI-TOF spectra of rat serum peptides prepared with ZipTip µc18 pipette tip out of (A) straight serum, (B) 50% acetonitrile supernatant and (C) serum processed by ultrafiltration. The last method provided stronger MALDI-TOF signal, higher signal-to-noise ratio and double the number of detected peptides. A B C Figure 6.1 MALDI-TOF spectra of (A) unprocessed rat serum; (B) serum peptides in 50% acetonitrile supernatant; and (C) serum ultrafiltrate processed with Amicon Ultra-4 30K NMWL centrifugal device. Figure 6.2 MALDI-TOF spectra of rat serum peptides after concentration and desalting by reversed phase chromatography: (A) rat serum processed with ZipTip µc18 pipette tip; (B) serum supernatant after 50% acetonitrile precipitation processed with ZipTip µc18 pipette tip; (C) 30K serum ultrafiltrate processed with ZipTip µc18 pipette tip; and (D) the same as (C) but 20% acetonitrile was added to the serum prior to ultrafiltration. A B C D 25

28 We have also investigated whether the addition of acetonitrile to serum prior to ultrafiltration improves the detection of serum peptides (MALDI spectrum shown in Figure 6.2D). Further improvement of the spectrum was attained, especially in the m/z 1000 area of the spectrum. Conclusion For the analysis of serum peptides, complexity reduction by eliminating higher molecular weight proteins is critical for high resolution mass spectrometry. Efficient separation of peptides from the majority of proteins and salts can be achieved by sample ultrafiltration. We have shown the effective use of Amicon Ultra-4 30K NMWL centrifugal devices for the preparation of peptides. Other molecular weight cut offs can be utilized depending on the desired range of peptides. The method can be used directly in combination with ZipTip µc18 pipette tips for peptide identification by MS/MS or as a first step prior to further surface-mediated enrichment using SELDI-TOF methods. References 1. Ardekani AM, Liotta LA, Petricoin III. Expert Rev Mol Diagn 2002;2: Carter D, Douglass JF, Cornellison CD, Retter MW, Johnson JC, Bennington AA, Fleming TP, Reed SG, Houghton RL, Diamond TS, Vedvick TS. Biochemistry 2002;41: Wellmann A, Wollscheid V, Lu H, Ma ZL, Albers P, Schutze K, Rohde V, Behrens P, Dreschers S, Ko Y, Wernert N. Int J Mol Med 2002;9: Petricoin EF, Ardekani AM, Hitt BA, Levine PJ, Fusaro VA, Steiberg SM, Mills GB, Simone C, Fishman DA, Kohn EC, Liotta LA. Lancet 2002; 359: Bischoff R, Luider TM. J Chrom B 2004;803: Stevens EV, Liotta LA, Kohn EC. J Gynecol Cancer 2003;13: Schulz-Knappe P, Schrader M, Standker L, Richter R, Hess R, Jurgens M, Forssmann W-G. J Chromatogr A 1997;776: Basso D, Valerio A, Seraglia R, Mazzza S, Piva MG, Greco E, Fogar P, Gall N, Pedrazzoli S, Tiengo A, and Plebani M. Pancreas 2002;24: Prazeres S, Santos MA, Ferreira HG, Sobrinho LG. Clin Endocrinol (Oxf) 2003;58: Tirumalai RS, Chan KC, Prieto DA, Issaq HJ, Conrads TP, Veenstra TD, Mol Cell Proteomics 2003;10:

29 Protocols Proteins Rapid Antibody Concentration with Amicon Ultra Centrifugal Filters Ultrafiltration offers a fast and convenient way to concentrate antibodies purified from serum, ascites fluid, or hybridoma supernatants. The traditional purification protocol for immunoglobulins includes affinity binding to Protein A or G chromatography media, washing unbound proteins, and eluting with a low ph buffer. Subsequently, purified antibodies are often too dilute for their intended purpose or for long-term storage. In addition, harsh elution conditions sometimes require buffer exchange to preserve protein activity. Dialysis is often used for antibody concentration and buffer exchange. However, ultrafiltration provides a quick, alternative method to concentrate and diafilter immunoglobulins with up to 99% recovery and one-step salt removal (see Concentration, Desalting, and Buffer Exchange with Amicon Ultra and Microcon Centrifugal Filters, page 16). To demonstrate the suitability of Amicon Ultra devices for concentrating purified IgG, rabbit serum was processed with Montage PROSEP-A and PROSEP-G Antibody Purification Kits (Millipore) and then concentrated with Amicon Ultra-15 30K NMWL devices. Figure 7.1 shows a decrease in the retentate volume proportional to the increase in antibody concentration. A twenty-minute centrifugation resulted in > 95% recovery of immunoglobulins. Figure 7.2 shows an SDS-PAGE gel of purified rabbit IgGs before and after ultrafiltration. IgG Volume (ml) PROSEP-A PROSEP-G Centrifugation Time (min) Figure 7.1 Concentration of rabbit IgG with Amicon Ultra-15 devices. The IgGs were purified using Montage PROSEP-A or PROSEP-G Antibody Purification Kits. The lines show IgG volume reduction, while the bars show a proportional increase in IgG concentration after 20 minutes of centrifugation time. Figure 7.2 SDS-PAGE gel of purified rabbit IgG before and after concentration with Amicon Ultra-15 devices. Lane 1: Lanes 2, 3: Lanes 4, 5: MW standards PROSEP-A-purified IgG before concentration (lane 2) and after concentration (lane 3) PROSEP-G-purified IgG before concentration (lane 4) and after concentration (lane 5) Protein Load: 5 µl in lanes 2 and 4; 1 µl in lanes 3 and 5 kda IgG Concentration (mg/ml) Heavy Chain Light Chain 27

30 Protocols Proteins Removal of Unincorporated Label from Labeled Protein with Amicon Ultra or Centricon Centrifugal Filters Introduction Centrifugal devices containing ultrafiltration membranes are ideal for the removal or exchange of salts, sugars, nucleotides, non-aqueous solvents, and other materials of low molecular weight. They also serve to separate free from bound species. One of the applications is removal of unincorporated label in protein labeling applications where Millipore centrifugal devices provide fast, convenient, high-recovery alternatives to gel filtration. Sample dilution, often associated with gel filtration, is not a problem. A few rounds of diafiltration can efficiently remove unincorporated label. Two examples below demonstrate the use of Millipore centrifugal ultrafiltration devices for removal of unreacted fluorescent and isotope labels. Method for Removal of FITC from FITC-BSA 1. Bovine serum albumin was labeled with fluorescein-isothiocyanate (FITC); the free unincorporated label was not removed. 2. Two ml of FITC-labeled BSA solution at 0.5 mg/ml were loaded into two Amicon Ultra-4 10K NMWL devices and centrifuged at 3,000 x g for 10 minutes. 3. Retentates (about 50 µl each) were re-diluted to 2 ml with water and centrifuged again. This step was repeated twice. 4. After each ultrafiltratation, the retentate and filtrate were analyzed by SDS-PAGE and their fluorescence measured on a SpectraFLUOR plate reader (Tecan) at excitation 485 nm and emission 530 nm. 5. The SDS PAGE gel was scanned on a Storm (GE Healthcare) fluorescence scanner. Results Figure 8.1 shows the SDS-PAGE gel of FITC-labeled BSA before and after each of four diafiltration cycles. Unincorporated FITC is clearly visible in the starting material and in the first filtrate. After only one cycle of ultrafiltration, the majority of the free label is removed. Subsequent cycles of filtration result in BSA that is virtually free of unincorporated FITC. Fluorescence measurement offers a more sensitive method to monitor the ultrafiltration process. Figure 8.2 shows the change in fluorescence of filtrate and retentate during four cycles of FITC-BSA diafiltration. The results indicate that almost 80% of free FITC can be removed after the first ultrafiltration and three rounds result in 98% removal. Therefore, ultrafiltration can be used to clean up protein labeling reactions as a viable alternative to gel filtration. Each ultrafiltration using Amicon Ultra-4 devices is accomplished in minutes and allows high recovery of target protein. While gel filtration results in diluted protein fractions, ultrafiltration offers the additional advantage of concentrating protein while removing unincorporated label. Purification of Radiolabeled Tumor Necrosis Factor Courtesy of Mathew L. Thakur, Ph.D., Thomas Jefferson University Hospital, Philadelphia, PA Tumor necrosis factor (TNF) is a low-mw protein (approximately 17 kda) predominantly derived from macrophages (TNF-alpha) or activated lymphocytes (TNF-beta). Different cell types, normal and malignant, possess receptors for TNF. The finding that TNF can destroy tumors in vivo, even in the absence of the direct lytic effect of neoplastic cells, has led researchers to the hypothesis that TNF 28

31 Figure 8.1 SDS-PAGE of FITC-labeled BSA 60,000 Lane 1: Lane 2: Lane 3: Lane 4: Lane 5: Lane 6: Lane 7: Lane 8: Starting material Retentate after first ultrafiltration Retentate after 2 rounds of ultrafiltration Retentate after 3 rounds of ultrafiltration Retentate after 4 rounds of ultrafiltration Filtrate after first ultrafiltration Filtrate after 2 rounds of ultrafiltration Filtrate after 3 rounds of ultrafiltration Fluorescence (Aribitrary Units) 50,000 40,000 30,000 20,000 10,000 0 Starting material Retentate Filtrate Spin 1 Spin 2 Spin 3 Spin 4 FITC-BSA Figure 8.2 Fluorescence measurements of the filtrates and retentates after each of four cycles of FITC-labeled BSA ultrafiltration. Free label was transferred through the 10,000 NMWL membrane while labeled BSA was retained and concentrated. All retentates and filtrates were volume adjusted to 2 ml prior to the measurement. FITC labeled with short-lived gamma-emitting radionuclides may be useful in scintigraphic imaging of certain tumors. In order to prepare radiolabeled TNF, ten micrograms of purified TNF were labeled with metastable isotope Tc-99m. Centricon 3K NMWL microconcentrators and Sephadex G-25 gel filtration columns were used to separate Tc-99m-TNF from unbound Tc-99m. Human serum albumin and lysozyme were added as a carrier in both cases. The results of this experiment indicate that Centricon 3K NMWL concentrators may be used to purify proteins of small molecular weight from unbound radioactivity without excessive loss of proteins. When the quantity of such a protein is limited, the addition of some carrier molecule after radiolabeling may be necessary. While eliminating more than 98% of the unbound radioactivity, Centricon 3K NMWL units concentrate the protein with high recovery and without loss, denaturation or excessive dilution. Table 8.1 Comparison of Centricon centrifugal device and gel filtration for purification of Tc-99m TNF Centricon Sephadex Device Column TNF Recovery 83 ± 2% 77.3 ± 8.5% Time 70 minutes 40 minutes Volume 75 µl 9 ml 29

32 Protocols Proteins Urine Concentration with Amicon Ultra Centrifugal Filters Introduction The measurement of specific proteins in urine is important for the diagnosis and management of disease states. In most cases, the content of these proteins in urine is too low to be detected and needs to be concentrated. Amicon Ultra-4 devices can be used to concentrate urine samples prior to clinical laboratory analyses. For example, patients with multiple myeloma exhibit a proliferation of one antibody-producing plasma cell, which leads to excess production of free immunoglobulin light chains known as Bence-Jones proteins. After sample enrichment in the Amicon Ultra-4 10K NMWL device, immunofixation electrophoresis can be used to identify free light chains (Bence-Jones proteins) in urine by forming a light chain-antibody complex. Also, agarose electrophoresis can be used to quantitate light chains and identify additional low molecular weight proteins such as albumin, α-1 globulins, transferrin and IgG that can be present in renal tubular disorders. Ultrafiltration of urine samples in Amicon Ultra-4 devices provides reproducible, high sample recovery for electrophoretic analyses, usually in 45 minutes or less. Materials Amicon Ultra-4 device, 4 ml, 10K NMWL Centrifuge with fixed-angle or swinging bucket rotor capable of 3400 x g Kit for microprotein determination (i.e. Sigma # 610-A/Brilliant blue G/Coomassie blue) Pipetter with 200 µl tip Electrophoresis (agarose gel) and immunofixation equipment with apparatus and reagents Method 1. Determine the total protein in a 24-hour urine specimen. 2. Fill Amicon Ultra-4 device with 4 ml of urine. 3. Centrifuge at 3400 x g for minutes (approximately µl concentrate volume). This produces up to a 160-fold increase in concentration. 4. Insert a pipetter into the bottom of the filter unit and withdraw the concentrated sample. 5. Perform agarose electrophoresis on the concentrate to quantitate light chains and identify other proteins. Determine the percentage of light chains with respect to the total number of components in the urine. Then multiply the percentage of light chains by the total 24-hour protein concentration (grams per 24-hour volume). 6. Perform immunofixation electrophoresis on the concentrate to identify light chains. Acknowledgements Research using Amicon Ultra devices for urine concentration in this protocol was conducted by Mark Merchant, Ph.D. at Helena Laboratories, Beaumont, TX. 30

33 References 1. Tietz N., Clinical Guide to Laboratory Tests, 2nd ed. Philadelphia:W.B. Saunders; 1990; Kahns L, Clinical Chemistry 1991; 37: Cleveland Clinic homepage. Accessed July myeloma/diagnosisandtreatmentof MultipleMyeloma.html 4. Christenson RH, et al. Clinical Chemistry 1983;29(6): Christenson RH, and Russell ME. Clinical Chemistry 1985;31(6):973. Additional Notes 1. Amicon Ultra devices can also be used to concentrate serum, plasma and cerebrospinal fluid for similar analyses. A concentration of approximately 20 mg/ml is required in order to detect free light chains from diseased patients by agarose electrophoresis. Detection by immunofixation electrophoresis is 10 times more sensitive than by agarose electrophoresis. 2. Normal heterogeneous immunoglobulins may also be seen in urine concentrate with immunofixation electrophoresis. This ladder effect is comprised of microheterogenous light chains. Bence-Jones proteins may be within this ladder. To verify the presence of Bence-Jones proteins requires additional analysis by two-dimensional electrophoresis. 3. If there is excess antigen, dilution of the concentrate will be required until equilibrium is achieved between the antigen (Bence-Jones protein) and the antibody. 4. Millipore also offers static concentrators (Minicon devices) for concentration of Bence-Jones protein in urine. 31

34 Protocols Proteins Affinity Purification with Ultrafree-MC Centrifugal Filters Introduction Affinity interaction chromatography is often the single most effective step in any protein purification procedure. Up to 95% purity can be achieved in one step, depending on the nature of the interaction and the starting composition of the protein solution. Well known examples of highly specific affinity interactions include antibodies and protein A/G; multiple histidine tags and nickel; streptavidin and biotin; antibodies and antigens; and many others. Less specific interactions are also used for enrichment or depletion protocols, including albumin depletion on cibacron blue resin, glycoprotein enrichment on concavalin A resin, and capture of nucleic acid-binding proteins on heparin resin. Affinity chromatography is often employed in the small-scale batch mode as a quick method for microgram-scale protein purification. The typical protocol involves: 1. Pipetting a small volume of affinity resin into a microfuge tube that contains the sample 2. Vortexing the tube for a few minutes 3. Centrifuging the resin to the bottom 4. Pipetting off the supernatant 5. Washing a few times (using steps 2 and 3) 6. Eluting with a small amount of eluant Figure 9.1 Ultrafree-MC centrifugal filter unit Although the method is relatively simple, care must be taken not to remove the chromatography resin when pipetting off the supernatant. Pre-packed mini-spin columns are a convenient tool for small-scale protein purification. They are operated by centrifugation and usually require less than an hour for the whole procedure. Another alternative for small-scale purification are centrifugal devices with microporous membrane, such as Ultrafree-MC centrifugal devices. The sample can be added to the filter basket and mixed for the needed residence time and then centrifuged. The process removes the interstitial liquid but does not dehydrate the beads. Washing and elution can also be performed in a similar manner and are more effective due to the efficient removal of buffer and/or eluant. Ultrafree-MC centrifugal filter units with microporous membrane (Figure 9.1) come with low protein-binding Durapore PVDF membrane in five different pore sizes from 0.1 to 5.0 µm. Affinity resin can be loaded into the filter basket and the device used as a home-made mini-spin column. We show the applicability of the device for purification of rabbit IgG on PROSEP-A resin and His-tagged C-RP protein on three different commercial metal-chelate resins. Materials Ultrafree-MC 0.45 µm centrifugal devices (Millipore cat. no. UFC3 0HV 00) PROSEP-A high capacity resin (Millipore cat. no ) Rabbit serum Gibco (Invitrogen lot no ) Micro-centrifuge Biofuge Pico (Heraeus instruments) Jouan CR1822 fixed angle rotor centrifuge 32

35 Xcell SureLock Mini-cell vertical electrophoresis system (Invitrogen cat. no. EI0001) NuPage NOVEX Bis-Tris 4 12%, 1 mm thick, 15 well SDS gels, (Invitrogen cat. no. NP0323) NuPage Sample Reducing agent (10X) (Invitrogen cat. no. NP009) NuPage SDS Sample Buffer (4X) (Invitrogen cat. no. NP007) SimplyBlue SafeStain Coomassie G-250 stain (Invitrogen cat. no. LC6060) Method for IgG Purification Solutions PROSEP-A binding buffer A: 1.5 M Glycine/NaOH, 3 M NaCl, ph 9.0 PROSEP-A elution buffer B2: 0.2 M Glycine/HCl, ph 2.5 PROSEP-A neutralization buffer: 1 M Tris/HCl, ph 9.0 Procedure mg of PROSEP-A media were placed in Ultrafree-MC 0.45 µm filter basket. 2. The columns were equilibrated with 400 µl of binding buffer A and centrifuged for 1 minute at 100 x g µl of rabbit serum were diluted 1:1 with binding buffer and the entire volume was loaded into the spin column containing PROSEP-A resin. 4. Devices were placed on a shaker for 15 minutes at room temperature and centrifuged at 100 x g for 5 minutes. Flow-through was collected for future analysis. 5. Three consecutive washes of 400 µl each were performed by adding 400 µl of binding buffer A and centrifuging at 2,000 x g for 2 minutes each µl of elution buffer B2 were added and centrifuged for 2 minutes at 2,000 x g µl of neutralization buffer were added to each collection tube. A second elution was collected after repeating the same process one more time. Method for His-tagged C-RP Purification Solutions Lysis buffer: 50 mm sodium phosphate, 300 mm sodium chloride,10 mm immidazole, ph 7 Binding buffer: 50 mm sodium phosphate, 300 mm sodium chloride, 10 mm immidazole, ph 7 Wash buffer: 50 mm sodium phosphate, 300 mm sodium chloride, 20 mm immidazole, ph 7 Elution buffer: 50 mm sodium phosphate, 300 mm sodium chloride, 250 mm immidazole, ph 7 1 mg/ml Lysozyme stock Benzonase Procedure 1. Recombinant proteins were expressed in Escherichia coli. 2. Cells were prepared at a 10X concentration using lysis buffer. Lysozyme was added to a concentration of 0.1 mg/ml. To reduce the viscosity, benzonase was added to the lysate. The lysates were clarified by centrifugation µl of the 50% resin slurry were added to the Ultrafree-MC device and the residual fluid was removed by centrifugation for 1 minute at 500 x g. 4. The resin was equilibrated with 500 µl of binding buffer and centrifuged for 2 minutes at 500 x g µl of the clarified lysate were added to the resin. 6. The His-tagged proteins were bound for 30 minutes with light agitation. 7. The lysate was removed by centrifugation at 500 x g for 10 minutes. 8. The resin was washed with 500 µl of wash buffer for 5 minutes with agitation. The wash solution was removed by centrifugation for 5 minutes at 500 x g. This step was repeated two more times µl of elution buffer were added to the Ultrafree-MC device and mixed for 5 minutes. Purified protein was recovered by centrifugation at 500 x g for 1 minute. 33

36 Figure 9.2 Rabbit IgG purification on PROSEP-A resin in Ultrafree-MC devices. Lane 1: Molecular weight standards Lane 2: Rabbit serum Lane 3: Flow through Lanes 4 6: Three consecutive washes Lanes 7, 8: Eluted IgG from two devices Figure 9.3 His-tagged protein purification using Ultrafree-MC devices. Lane 1: Molecular weight standards Lane 2: E. coli lysate expressing C-RP protein Lane 3: E. coli lysate expressing RT66 protein Lanes 4, 5: Proteins purified on Ni-NTA resin Lanes 6, 7: Proteins purified on BD Talon resin kda kda RT66 C-RP Results The results of purifying rabbit IgG using Ultrafree-MC centrifugal devices are shown in Figure 9.2. The device was challenged with approximately 14 mg of total protein, with an estimated IgG content of mg. The original serum, flow-through, three washes and two eluted fractions were analyzed by SDS-PAGE. The total amount of purified IgG, as estimated by OD 280, was 1.2 mg and 1.1 mg on two devices processed in parallel. The whole procedure was completed in less than 1 hour. This method can be useful for monitoring the titer of antigen-specific antibodies after immune activation, or whenever small amounts of IgG need to be purified. Figure 9.3 shows the results of His-tagged protein purification in Ultrafree-MC devices using Ni-NTA agarose and BD Talon resin. Two recombinant proteins were purified: C-RP, highexpressing protein of 26 kda; and RT66, lowexpressing protein of 66 kda. As seen in Figure 9.3, both purifications resulted in high purity proteins. The amount of proteins purified out of 500 µl of lysate was µg for C-RP protein and 8 µg for RT66. The data show that affinity batch purification can be effectively performed on a small scale using Ultrafree-MC devices loaded with resin. The process combines the high efficiency of batch binding and washing with the handling convenience of a mini-spin column. With minimal hands-on time, the method provides flexibility of resin to lysate ratio and binding conditions, independent of centrifugation speed and rotor angle. This method is applicable to recombinant protein purification, antibody purification and immunoprecipitation. 34

37 Protocols Proteins Use of Centrifugal Filter Devices as an Alternative to Stirred Cells Introduction Stirred cell devices have been successfully used to purify proteins from large volumes of solution for many years. However, the need for assembly and cleaning between uses, and the lengthy separation times, can present difficulties for active, or understaffed, laboratories. Millipore s large volume centrifugal filter devices come preassembled and ready to use. Individual devices are available for processing 20 or 70 ml volumes. For larger volumes, multiple devices can be spun simultaneously. Spin times are typically measured in minutes. And unlike stirred cells, centrifugal devices run unattended. There is no need to refill a reservoir and, therefore, less risk of adventitious contamination. The following protocol demonstrates the use of Centricon Plus-70 centrifugal filter devices to purify and concentrate proteins from large volumes of solution. Abstract This study aims to achieve purification and concentration of a fusion protein composed of the alpha and gamma subunits of the human high affinity IgE receptor. The alpha-gamma fusion protein is purified and then coupled to Sepharose (GE) beads to produce an affinity column for isolating human IgE antibodies. Method The alpha and gamma subunit components of the high affinity IgE receptor were transfected into mouse myeloma cell line NS0 and the secreted fusion protein was purified on a Protein G column. The eluted proteins were concentrated from volumes of ml to 5 10 ml using a Centricon Plus-70 centrifugal filter device in a swinging bucket rotor at 3000 x g for 10 minutes (60 ml per filter unit). Buffer exchange was also performed subsequently using the same units in tissue culture grade sodium azide-free PBS. The concentrated samples were then filter-sterilized under sterile conditions using a 0.2 µm pore filter. Results Spectrophotometric evaluation of alpha-gamma fusion protein recovery using Centricon Plus-70 devices showed a result of 18.8 mg/l of supernatant. HPLC trace analysis revealed the chromatogram shown in Figure Acknowledgement Protocol courtesy of GKT School of Biomedical Sciences, London. Absorbance Units Time (min) Figure 10.1 HPLC profile of alpha-gamma fusion component of the human FcεRI 35

38 Protocols Nucleic Acids Concentrating and Desalting DNA or RNA with Microcon or Centricon Centrifugal Filters Introduction Centrifugal filter devices serve as powerful tools in molecular biology applications, such as DNA or RNA concentration and desalting procedures. Ultrafiltration (UF) is a pressure-driven, convective process that uses semipermeable membranes to separate species by molecular size and shape. UF is highly efficient, allowing for concentration and desalting at the same time. Unlike the use of chemical precipitation methodologies (i.e., ethanol, phenol/chloroform), there is no phase change, Table 11.1 Nucleotide cut-off guidelines for Microcon and Centricon centrifugal devices (based on >90% recovery of nucleic acids) Single-Stranded* Double-Stranded NMWL Nucleotide Cut-off (bp) Nucleotide Cut-off (bp) 3K K K K K *Single-stranded nucleic acids with extensive secondary structure will be better retained than those without. Table 11.2 Desalting and recovery with Centricon 30K NMWL devices Spin Number % DNA Recovered % CsCl Removed Using 1 ml of 25 µg/ml E. coli DNA in 6 M CsCl, sample was repeatedly concentrated to 0.1 ml. Sample spun at 2000 x g in a 45 degree fixed angle rotor. The concentrated sample was reconstituted to original 1 ml volume by adding 50 mm Tris. After three spins, CsCl concentration was reduced by four orders of magnitude. Equivalent results can be obtained with the Microcon 30K NMWL filter unit (as well as other NMWLs). which often denatures labile species. DNA and RNA samples with starting concentrations as low as 5 ng/ml can be routinely concentrated in minutes with 99% recovery of starting material, and without the use of co-precipitants. Centrifugal concentrator devices are ideal for separating high and lowmolecular weight species. Ultrafiltration can also be used to change solvents by diafiltration. In this process, the sample is concentrated, then diluted to the original volume with the desired buffer and concentrated again, thus washing out the original solvent. Millipore ultrafiltration membranes are characterized with well-defined, globular solutes (proteins). Typically, the nominal molecular weight limit (NMWL) for a membrane is the point at which over 90% of a solute with that molecular weight will be retained. To process DNA or RNA, the membrane needs to be characterized according to the number of nucleotides in the fragment. Polynucleotides (DNA and RNA) have tertiary structures that are ordinarily more extended than those of typical globular proteins of similar size. Millipore has determined nucleotide cut-offs (based on the number of bases or base pairs in a fragment of DNA or RNA) that correspond to the NMWL of each of their low binding membranes. The nucleotide cut-off (NCO) indicates the fragment length of single- or doublestranded DNA or RNA that one would expect to recover at 90% efficiency with a unit of the named NCO. It is best to choose the NCO with about half the length of the fragment of interest. For example, selecting a 30K NMWL membrane for a 50 base pair fragment generally results in 90% product recovery. A 10K NMWL membrane would provide for closer to 100% recoveries but would take much longer to process the sample. For nucleic acid samples >500 base pairs, a 100K NMWL membrane is appropriate. 36

39 Table 11.1 offers guidelines for DNA/RNA retention based on the nucleotide content of singleand double-stranded pieces. For example, more than 90% of a single-stranded 30-mer will typically be retained by a Microcon 10K NMWL or a Centricon 10K NMWL centrifugal filter device. Concentrating dilute DNA solutions is a key step for many subsequent preparative and analytical procedures. For example, standard plasmid preparations involving cesium chloride, equilibrium centrifugation, and gel filtration yield DNA in large volumes that require concentrating prior to precipitation. DNA concentration is also necessary in purifying restriction fragments from gels. There are three major techniques currently available for concentrating nucleic acids: repeated extractions with n-butanol adsorption to ion exchange resin, followed by high salt elution lyophilization The first method has the disadvantage that n-butanol concentrates all solutes, including salt, which tends to co-precipitate with DNA upon addition of ethanol. The second method, ion exchange, aside from requiring buffers of various ionic strengths, yields DNA in a high salt solution. The third method, lyophilization, increases the concentration of buffer components, which can result in degradation of nucleic acids. Ultrafiltration membranes retain DNA or RNA but are permeable by smaller ionic buffer components. Ultrafiltration alone does not change buffer composition. The salt concentration in a sample concentrated by Microcon or Centricon centrifugal filter devices will be the same as in the original sample. For desalting, the concentrated sample is diluted with water or buffer to its original volume and spun again in a process called diafiltration. This removes the salt by the concentration factor of the ultrafiltration. For example, if a 500 µl sample containing 100 mm salt is concentrated to 25 µl (20X concentration factor), 95% of the total salt in the sample will be removed. The salt concentration in the sample will remain at 100 mm. Rediluting the sample to 500 µl will bring the salt concentration to 5 mm. Concentrating to 25 µl once more will remove 99% of the original total salt. The concentrated sample will now be in 5 mm salt. For more complete salt removal, an additional redilution and spinning cycle will remove 99.9% of the initial salt content (see Table 11.2). Figure 11.1 shows that both recovery and biological activity are similar when DNA is concentrated with Millipore s high recovery centrifugal filter devices versus conventional techniques. However, the efficiency and immediate capability to further utilize the nucleic acids without a subsequent desalting step is a significant advantage when using Millipore centrifugal devices. Methods Microcon Device 1. Select a Microcon unit with nucleotide cut-off equal to or smaller than the molecular size of the nucleic acid you want to retain (refer to Table 11.1). 2. Insert Microcon sample reservoir into one of the two vials provided for each unit. 3. To concentrate (without affecting salt concentration): Pipette up to 500 µl of DNA or RNA sample into the reservoir. Spin for recommended time, not exceeding recommended g-force guidelines shown in Table Table 11.3 Recommended g-force and spin time for Microcon devices Maximum Spin Time Spin Time G-Force (min.) (min.) NMWL Rating at 4 C at 25 C 3K 14, K 14, K 14, K 14, K Figure 11.1 Retention of labeled DNA fragments by Centricon 10K and 30K NMWL devices. Two 1 µl aliquots of 32 P-labeled pbr322 DNA fragments were diluted to 1.0 ml using TE buffer (10 mm Tris-HCl, 1 mm EDTA, ph 8.0). They were subsequently concentrated to 40 µl with the Centricon devices. 20 µl of each concentrate were analyzed on a 2.5% agarose gel. The intensities of the autoradiographic bands were compared to bands from a 1 µl aliquot of the solution of labeled DNA fragments that had not been concentrated by the devices. The gel was dried and autoradiographed. Lane 1: Not concentrated using Centricon device Lane 2: Concentrated with Centricon 10K NMWL device Lane 3: Concentrated with Centricon 30K NMWL device / /

40 4. To exchange salt: Add the proper amount of appropriate the diluent to bring the concentrated sample to 500 µl. Spin for the recommended time, not exceeding g-force shown in Table To achieve lower salt concentration, repeat the entire step as necessary. NOTE: Do not let filtrate vial overfill. 5. Remove reservoir from vial and invert into a new vial (save filtrate until sample has been analyzed). 6. Spin for 2 minutes at x g to recover nucleic acid in the vial. 7. Remove reservoir. Cap vial to store. Table 11.4 Recommended g-force and spin time for Centricon devices Maximum Spin Time (min.) NMWL G-Force Rating at 25 C 3K 7, K 5, K 5, K 5, K 1, Centricon Device 1. Select a Centricon unit with a nucleotide cut-off equal to or smaller than the molecular size of the nucleic acid you want to retain (refer to Table 11.1). 2. To concentrate (without affecting salt concentration): Pipette up to 2 ml of DNA or RNA sample into the reservoir. Spin for the recommended time, not exceeding g-force shown in Table To exchange salt: Add the proper amount of appropriate the diluent to bring the concentrated sample to 2 ml. Spin for the recommended time, not exceeding g-force shown in the Table To achieve lower salt concentration, repeat the entire step as necessary. NOTE: Do not let filtrate vial overfill. 4. Remove reservoir from filtrate vial and invert unit (save filtrate until sample has been analyzed). 5. Spin for 2 minutes at x g to recover nucleic acid in the retentate vial. 6. Remove reservoir. Cap retentate vial to store. 38

41 Protocols Nucleic Acids Preparing Samples for Forensics Identification Analysis with Microcon Centrifugal Filters Centrifugal filter devices with ultrafiltration (UF) membranes are used by forensics laboratories to isolate genomic DNA for human identification. The source materials are typically blood stains and/or other bodily fluids obtained from crime scenes, criminal suspects, or human remains. The isolated genomic DNA is used to identify individual persons based on their patterns of Short Tandem Repeats. STRs are polymorphic DNA loci that contain repeated DNA sequences. Because the repeats can vary from two to seven bases in length, many different alleles are possible for each locus. The parallel analysis of 9 to 13 polymorphic STR loci can unequivocally identify an individual. Several suppliers offer STR assay kits. Each supplier offers several versions of their kits with different protocols, modes of operation, and detection systems to suite the user s needs. Millipore s UF-based centrifugal devices are specified in several of these protocols (AmpFLSTR Profiler, Applied Biosystems; GenePrint STR and GenePrint Fluorescent STR Systems, Promega). Use of UF-based Centrifugal Devices in New York City Depending on the type of evidence under investigation, the Department of Forensic Biology in New York City had been preparing genomic DNA by either (1) organic extraction (phenol/chloroform/ isoamylalcohol) followed by alcohol precipitation or (2) direct lysis in the presence of Chelex beads (Bio-Rad) with no further purification. The Department of Forensic Biology s laboratory has replaced the ethanol precipitation step with Microcon centrifugal filters with a 100K NMWL ultrafiltration membrane. The centrifugal devices provide a faster way to concentrate the purified DNA without the use of toxic chemicals. The laboratory also uses the Microcon 100K NMWL device after Chelex extraction to remove inhibitors that can adversely affect PCR amplification. In addition, Chelex extraction of small bloodstain samples often results in DNA concentrations below detection limits. Concentrating extracted samples with Microcon 100K NMWL devices improves the success rate of STR amplification and yields a full STR allele profile even with minute blood or semen stains. According to the New York City Department of Forensic Biology, several of the peaks shown in Figure 12.1 would have been below the detection threshold and identification would have been negative or inconclusive without sample cleanup using Microcon devices. A B Figure 12.1 Genotype analysis of a semen sample before and after concentration by a Microcon centrifugal filter. Electropherogram A shows a partial profile with a high molecular weight loci in each color missing. Electropherogram B shows a full profile and higher peak heights. Amounts amplified were 0.62 ng for electropherogram A and 1 ng for electropherogram B. The sample was extracted with Chelex beads. The peaks are labeled with allele name, size in base pairs, and fluorescent peak height. The improvement in results is due not only to the increased DNA input but also to the concentration step following extraction. 39

42 Protocols Nucleic Acids PCR Purification with Montage PCR Filter Units Introduction After polymerase chain reaction (PCR) 1, amplified DNA must be separated from excess reaction components that can interfere with subsequent manipulations such as cloning or sequencing. The Montage PCR filter unit is a single-use device that simplifies the purification of PCR products. It concentrates amplified DNA and removes primers and unincorporated dntps, while providing excellent capacity, high recovery, and high purity. The method is quick and highly reproducible, which makes it ideal for processing one-up or multiple samples in middle-throughput operations. Materials Variable speed microcentrifuge Montage PCR filter unit Purified water (such as Milli-Q water) or TE buffer PCR reaction (aqueous phase) Procedure 1. Insert the Montage PCR sample reservoir into one of the two vials provided. 2. Fill the reservoir with 300 µl purified water or TE buffer. Add 100 µl PCR reaction to the reservoir (step 1). Smaller volumes of PCR product may be used, but the volume should be adjusted to a final volume of 400 µl. 3. Spin the Montage PCR device at 1000 x g for 15 minutes (step 2). NOTE: For optimal recovery, do not centrifuge longer than the specified 15 minutes or greater than 1000 x g. 4. To recover DNA, remove the sample reservoir from filtrate collection vial and place in a clean vial. Primer Salt 5. Add 20 µl purified water or TE buffer to sample reservoir. 6. Invert the reservoir and spin at 1000 x g for 2 minutes to retrieve the purified PCR (step 3). Results DNA dntp Montage PCR filter units were used to purify 100 µl PCR reactions according to the specified protocol. After recovering the DNA fragments (n = 10) using a reverse spin, the samples were separated by agarose gel electrophoresis. Recoveries of the various PCR products were determined by densitometry (Figure 13.1). The Montage PCR filter unit is a convenient method for single-sample PCR purification. The high-performance device purifies PCR products in a single centrifugation step. Purified samples are ready for downstream applications with no additional purification steps. References Step 1 Step 2 Step 3 1. PCR is covered by U.S. patents issued to Hoffmann-LaRoche, Inc. 40

43 100% 80% Percent Recovery 60% 40% 20% 0% 137 bp 301 bp 500 bp 657 bp 1159 bp Figure 13.1 Purification of PCR products using Montage PCR Filter Units Figure 13.2 Typical electropherogram shows an 1159 bp PCR product purified with a Montage PCR filter unit. Note the uniform signal intensity and long read lengths. Purified samples are ready for cloning or sequencing with no additional purification steps. 41

44 Protocols Nucleic Acids Preparation of Fluorescent DNA Probe from Human mrna or Total RNA with Microcon Centrifugal Filters Introduction Microarrays are efficient tools that enable the high throughput identification of genes that are differentially regulated in response to disease, drugs or other stimuli. With the completion of several key genome sequencing projects, scientists now have the ability to custom design DNA microarrays specific to their research interests. The recent advances in robotics, bioinformatics and detection technologies have greatly simplified the manufacture and analysis of microarrays. However, the successful application of microarray technology requires highly purified, fluorescently labeled cdna probes. The application of ultrafiltration technology to this challenge has resulted in a robust, efficient and rapid method for the generation of high quality fluorescently labeled probes suitable for use with microarrays. The protocol below describes a method for the generation and purification of fluorescently labeled probes using a Microcon 30K NMWL centrifugal filter device. Method 1. To anneal primer, mix 2 µg of mrna or µg total RNA with 4 µg of a regular or anchored oligo-dt primer in a total volume of 15.4 µl as shown in Table Heat to 65 C for 10 minutes and cool on ice. 3. Add 14.6 µl of reaction mixture each to Cy3 and Cy5 reactions as shown in Table Incubate at 42 C for 1 hour. 5. Add 1 µl SSII (RT booster) to each sample. Incubate for an additional hours. 6. Degrade RNA and stop reaction by addition 15 µl of 0.1 N NaOH, 2 mm EDTA and incubate at C for 10 minutes. If starting with total RNA, degrade for 30 minutes instead of 10 minutes. 7. Neutralize by addition of 15 µl of 0.1N HCl. 8. Add 380 µl of TE (10 mm Tris, 1 mm EDTA) to a Microcon 30K NMWL device column. Next add the 60 µl of Cy5 probe and the 60 µl of Cy3 probe to the same Microcon device. NOTE: If re-purification of Cy dye flow-through is desired, do not combine probes until Wash Wash 1: Spin column for 7 8 minutes at 14,000 x g. 10. Wash 2: Remove flow-through and add 450 µl TE and spin for 7 8 minutes at 14,000 x g. It is a good idea to save the flow trough for each set of reactions in a separate microcentrifuge tube. 42

45 11. Wash 3: Remove flow-through and add 450 µl 1X TE, 20 µg of Cot1 human DNA (20 µg/µl, Gibco-BRL), 20 µg polya RNA (10 µg/µl, Sigma, #P9403) and 20 µg trna (10 µg/µl, Gibco-BRL, # ). Spin 7 10 min. at 14,000 x g. Look for concentration of the probe in the Microcon device. The probe usually has a purple color at this point. Concentrate to a volume of less than or equal to the volume listed in the Probe and TE column in Table These low volumes are attained after the center of the membrane is dry and the probe forms a ring of liquid at the edges of the membrane. Make sure not to dry the membrane completely. 12. Invert the Microcon device into a clean tube and spin briefly at 14,000 RPM to recover the probe. 13. Select the appropriate row from Table Adjust the probe volume to the value indicated in the Probe and TE column. 14. For final probe preparation add 4.25 µl 20X SSC and 0.75 µl 10% SDS. When adding the SDS, be sure to wipe the pipette tip with clean, gloved fingers to remove excess SDS. Avoid introducing bubbles and never vortex after adding SDS. The probe is now ready for hybridization. Acknowledgements Protocol courtesy of Patrick Brown, Max Diehn, and Ash Alizadeh, Stanford University School of Medicine Table 14.1 Preparation of primer Primer Cy3 Cy5 Notes mrna (1 µg/µl) x µl y µl 2 µg of each if mrna; µg if total RNA Oligo-dT (4 µg/µl) 1 µl 1 µl Anchored: 5'-TTT TTT TTT TTT TTT TTT TTV N-3' Purified H 2 O (DEPC) to 15.4 µl to 15.4 µl Total volume 15.4 µl 15.4 µl Table 14.2 Preparation of probe Final Reaction Mixture Volume Unlabeled dntps Volume Concentration 5X first-strand buffer* 6.0 µl datp (100 mm) 25 µl 25 mm 0.1 M DTT 3.0 µl dctp (100 mm) 25 µl 25 mm Unlabeled dntps 0.6 µl dgtp (100 mm) 25 µl 25 mm Cy3 or Cy5 (1 mm, Amersham) 3.0 µl dttp (100 mm) 10 µl 10 mm Superscript II (200 U/µL, Gibco-BRL) 2.0 µl Purified H 2 O 15 µl Total volume 14.6 µl Total volume 100 µl *5X first-strand buffer: 250 mm Tris-HCl (ph 8.3), 375 mm KCl, 15 mm MgCl 2 Table 14.3 Final probe preparation Cover Slip Size Total Hybrid Probe and TE 20X SSC* 10% SDS (mm) Volume (µl) (µl) (µl) (µl) 22 x x x *20X SSC: 3.0 M NaCl, 300 mm NaCitrate (ph 7.0) 43

46 Protocols Nucleic Acids Purification of In Vitro Synthesized mrna with Microcon or Centricon Centrifugal Filters Introduction In vitro transcription reactions employing T3, T7 or SP6 phage-encoded RNA polymerases are widely used to synthesize RNA from recombinant vectors containing appropriate promoters. Production of large amounts of specific RNA is valuable in the preparation of hybridization probes and in vitro translation studies; in the synthesis of ribozymes, rrna, SRP, antisense RNA and substrates for RNA splicing; and in RNA-protein interaction studies. Centricon and Microcon centrifugal filters are well suited for the purification of radiolabeled RNA transcripts 1. Ultrafiltration can simultaneously and efficiently remove unincorporated ribonucleotides and salts from the transcripts and concentrate the RNA. RNA molecules retain their integrity and are recovered with high yields. Purity of a transcript is especially important when it is used in in vitro translation systems. Trace amounts of ethanol, phenol, salts or excess cap analog used during the synthesis of capped mrna can cause a dramatic decrease in translation efficiency. After the transcription reaction is complete, template DNA is usually degraded by the addition of DNase I. The RNA is purified by two phenol/ chloroform extractions followed by ethanol precipitation. Other, less popular methods are gel purification (used predominantly when separation of full-length transcript from shorter RNAs is important, e.g., ribonuclease protection assays) or LiCl precipitation. A series of experiments was performed in our laboratory to determine the effectiveness of using Centricon and Microcon devices to purify in vitro synthesized mrna and in vitro translation studies. Results indicate that ultrafiltration can efficiently remove inhibitory contaminants from mrna preparations, leading to increased translational efficiencies. Methods RNA Transcription For our studies we chose plasmid pgem-luc containing the luciferase gene (luc) in the center of a multiple cloning cassette of the pgem-11zf (-) plasmid (Promega). DNA template was linearized with XhoI, followed by enzyme and salt removal by diafiltration in Microcon 100K NMWL devices. Linearized template was transcribed, using MEGAscript kit (Ambion) according to the recommended protocol. After the reaction was completed (3 to 4 hours), template DNA was degraded with DNase I and the reaction mix added to a Microcon 30K NMWL device filled with 450 µl of water. The device was spun for 20 minutes at 12,000 x g in a temperature-controlled centrifuge at 4 C. Purified, concentrated RNA was recovered by inverted spin. For the preparation of capped transcript, cap analog m7g (5 ) ppp (5 ) G (New England Biolabs, Inc.) was included in the transcription reaction and the level of GTP reduced (4:1 ratio of cap analog to GTP). To purify the transcript by phenol/chloroform extraction, the reaction mix was diluted with water and a one-tenth volume of ammonium acetate stop solution was added. The mixture was extracted once with phenol/chloroform, followed by chloroform extraction. RNA was precipitated with isopropanol and the pellet resuspended in distilled water. Alternatively, LiCl precipitation solution (one-half volume) was added to the reaction mix, followed by incubation at minus 20 C for 1 hour. RNA was pelleted by centrifugation and dissolved in water. Size and integrity of the in vitro transcription products were assessed by running an aliquot of the purified RNA transcript on a formaldehyde/formamide agarose gel. Ethidium bromide was added to the RNA before lading on the gel to stain the RNA sample and keep background fluorescence low 2. 44

47 Translation In Vitro In vitro translations were performed in the Flexi Rabbit Reticulate Lysate System (Promega) according to standard luciferase RNA translation conditions with minor modifications (Rnasin Ribonuclease inhibitor was omitted and 35 S-methionine added). Results of translation were analyzed by determination of percent incorporation of 35 S-methionine and fold stimulation, compared to controls without RNA. Minimum acceptable stimulation was 8-fold. Results Aliquots of RNA transcript purified by different methods (ultrafiltration, phenol extraction and LiCl precipitation) were run on a denaturing agarose/formaldehyde gel. Results are shown in Figure The banding pattern of the 1.7kb RNA transcripts is identical regardless of purification method. Similar results were obtained in the case of capped transcript (results not shown). The effect of increasing the mrna concentration on the translational efficiencies was examined. At low mrna levels, the capped luc mrna was translated three times more efficiently than the uncapped mrna (Figure 15.2). At higher mrna levels, the translation of both transcripts was comparable. Similar behavior was observed with CAT mrna 3. Even relatively high levels of mrna did not cause the decrease in translational efficiencies noted by other groups 4. This result could be attributed in part to the lack of inhibitory contaminants in the mrna preparation. We also checked the effect of the RNA clean-up method on in vitro translational efficiency. For details of various procedures, see the Methods section. RNA purified by each of the methods (1 µg) was translated and results showing total 35 S-methionine incorporation are presented in Table While there were no observable differences between these RNAs by gel electrophoresis analysis (Figure 15.1), RNA purified by ultrafiltration gave twice the translation efficiencies of phenol-extracted RNA. References 1. Krowczynska AM. biosolutions 1993;2(1): Ogretmen B, Ratajczak H, Kats A, Stark BC. Biotechniques 1993;14(6): Polayes D. Focus 1991;13(4): Dasso MC, Jackson RJ. Nucl. Acid. Res. 1989;17:3129. Figure 15.1 Comparison of pgem-luc transcript purification methods. Transcripts were synthesized in 20 µl reactions. After DNase I treatment, RNA was purified from the reaction mix. 500 ng of purified RNA were run on 1% agarose/formaldehyde gel. Lane 1: kb RNA ladder (Gibco BRL) Lane 2: RNA purified in Microcon 30K NMWL device Lane 3: RNA purified in Centricon 100K NMWL device Lane 4: RNA purified by phenol extraction Lane 5: RNA purified by LiCl precipitation Figure 15.2 Effect of pgem-luc RNA concentration on in vitro translation. Increasing amounts of uncapped Luc RNA transcripts and capped Luc RNA were used in translation reactions. Incorporation of 35 S-methionine was determined by TCA precipitation. Both RNA transcripts were purified with Microcon 30K NMWL devices. cpm x Capped pgem-luc RNA pgem-luc RNA µg RNA/50 µl Reaction Table 15.1 Effects of RNA clean-up method on translation efficiency RNA Total 35 S-methionine Incorporation RNA 1 972,000 RNA 2 514,000 RNA 3 776,000 The RNA transcripts were cleaned using DNase I incubation followed by purification in Centricon 100K NMWL devices (RNA 1), phenol/chloroform extraction (RNA 2), or precipitation with 7.5 M LiCl (RNA 3). 1 µg of each RNA was used to program 50 µl in vitro translation reaction, using the Flexi Rabbit Reticulocyte Lysate System. 35 S-methionine incorporation was determined by TCA precipitation. Data show the average of three independent experiments. 45

48 Protocols Nucleic Acids Quantitative Recoveries of Nanogram Amounts of Nucleic Acids with Microcon Centrifugal Filters Introduction Molecular cloning experiments often require concentration of nanogram quantities of DNA. When dealing with such small amounts of nucleic acids, the use of coprecipitants (trna or purified glycogen) is essential for effective DNA recovery 1. However, when carrier trna cannot be used (for example, when DNA is to be labeled with 32 P datp and polynucleotide kinase), the only method of recovering DNA as a precipitate in ethanol has been the ultracentrifugation method developed by Shapiro 2. Ultrafiltration, which has been traditionally used to concentrate and desalt protein samples simultaneously, can be an efficient alternative to ethanol precipitation. In this experiment, three popular ethanol precipitation protocols for DNA and oligonucleotides are compared with ultrafiltration in Centricon and Microcon centrifugal filter devices. For a detailed discussion on the effects of incubation time, temperature and centrifugation parameters on ethanol precipitation, see Reference 3. Methods Plasmid pbr322 was digested with EcoR I and the 3 recessed termini were filled-in with alpha 32 P datp, using Klenow fragment of DNA polymerase I. A 25 nucleotide mixed base oligomer was radiolabeled by phosphorylation with bacteriophage T4 polynucleotide kinase. Ethanol Precipitation All DNA precipitations were performed in 250 µl volume. Each tube contained a given amount of DNA, supplemented with 10 ng of radioactively labeled pbr322 in 0.3 M sodium acetate buffer. To precipitate the DNA, 2.5 volumes of 95% EtOH were added to each tube. The content was well mixed and incubated for the specified period of time at 20 C or 70 C (dry ice). The solutions were then centrifuged at 12,000 x g in a fixedangle microcentrifuge at 4 C for 15 minutes. The supernatants were carefully removed and pellets resuspended in 50 µl of TE buffer. The radioactivity in the precipitates and supernatants was then determined by counting Cherenkov radiation. The percentage of recovered DNA was calculated from precipitates and the initial counts. Each data point in the tables represents the average of at least three samples. To precipitate the oligomer, 60 µl of TE buffer containing a given amount of 25-mer and 5 ng of radiolabeled tracer was supplemented with 240 µl of 5 M ammonium acetate and 750 µl of EtOH. Subsequent steps were identical to those described for DNA. Ultrafiltration A solution of DNA (500 µl) was placed into a Microcon 30K NMWL unit and spun at 12,000 x g for 10 minutes. A 500 µl solution of oligonucleotides in TE buffer was placed into a Microcon 3K NMWL unit and spun for 45 minutes at 12,000 x g. The retentates were recovered by inverting the units and centrifuging at x g for 2 minutes. While concentrating the oligomer samples, it is important to avoid high salt concentrations which promote binding of single-stranded nucleic acids to the cellulose-based ultrafiltration membrane. The radioactivity in the retentates and filtrates was determined by counting Cherenkov radiation. Data represent averages of six samples each. 46

49 Results Standard methods for ethanol precipitation of nucleic acids and ultrafiltration are compared in Tables 16.1 and Use of Centricon 30K NMWL and Centricon 3K NMWL (data not shown) devices gave results similar to those obtained with Microcon devices. Recovery of DNA was assessed at close to 100%, indicating that no significant amount of nucleic acid was lost to the membrane or device due to adsorption. In contrast, DNA recovery using EtOH precipitation varied from as little as 14% as in the case of DNA (10 ng/ml) precipitated for 15 minutes at 70 C to a maximum of 76% after overnight incubation at 20 C. Discussion Poor recovery of nucleic acids at very low concentration with ethanol precipitation may be partially due to the fact that small amounts of DNA do not adhere well to the tube walls following sedimentation unless high g forces (ultracentrifugation) are employed. The yield of DNA incubated at 70 C is slightly reduced, in agreement with previous studies. Also an overnight precipitation at 20 C can significantly improve recovery. In contrast to EtOH precipitation, ultrafiltration using Microcon or Centricon devices delivered almost 100% recovery with concentrations as low as 10 ng/ml and took only 10 minutes. The ultrafiltration devices were found to concentrate and desalt nucleic acids effectively in one step resulting in high recoveries and providing a quick, alternative to ethanol precipitation. References 1. Wallace DM. Precipitation of Nucleic Acids. Methods in Enzymology 1987;152: Shapiro DJ. Quantitative Ethanol Precipitation of Nanogram Quantities of DNA and RNA. Anal Biochem 1981;110: Crouse J, Amorese D. Ethanol Precipitation: Ammonium Acetate as an Alternative to Sodium Acetate. Focus 1987;9(2):3 5. Table 16.1 Efficiency of different methods for concentration of DNA DNA Concentration 10 ng/ml 25 ng/ml 50 ng/ml 250 ng/ml 1000 ng/ml Method % Recovery % Recovery % Recovery % Recovery % Recovery EtOH, 70 C, 15 min EtOH, 20 C, 30 min EtOH, 20 C, 18 hr Microcon 30K NMWL Device Table 16.2 Efficiency of different methods for concentration of oligonucleotides Oligomer Concentration 10 ng/ml 25 ng/ml 50 ng/ml 250 ng/ml 1000 ng/ml Method % Recovery % Recovery % Recovery % Recovery % Recovery EtOH, 70 C, 15 min EtOH, 20 C, 30 min EtOH, 20 C, 18 hr Microcon 3K NMWL Device

50 Protocols Nucleic Acids Effect of Centrifugal Ultrafiltration on Large Fragment DNA Integrity Introduction Figure 17.1 Supercoiled DNA Ladder. Lane 1: Starting material Lanes 2, 3: Retentate from DNA ladder spun 3 times at 12,000 x g in Microcon 30K NMWL device Lane 4: Retentate from DNA ladder spun 3 times at 5,000 x g in Centricon 30K NMWL device Centrifugal ultrafiltration provides a fast and easy method for the concentration and desalting of biological molecules. Previous reports document the use of ultrafiltration for desalting samples of nucleic acids 1, removing excess primers from PCR 2 reactions 3, or concentrating DNA/RNA samples without the need to use ethanol precipitation 4. Centricon and Microcon filters offer a convenient, reproducible means for centrifugal ultrafiltration of samples from 50 µl to 2 ml. They insure high sample recovery with their patented inverted recovery spin. The analysis of complex genomes depends on the ability of the researcher to prepare pure, high molecular weight DNA. When preparing high molecular weight DNA samples for cosmid cloning or other applications, it is important to treat the DNA gently to avoid shearing or other damage to the sample. As virtually all protocols for the preparation of high molecular weight DNA require, at some point, buffer exchange and/or concentration, centrifugal ultrafiltration can provide an efficient alternative to standard procedures To be useful, centrifugation must not cause breakage of the DNA during the ultrafiltration procedure. This article summarizes the results of a series of experiments designed to evaluate the effect of g forces on various samples of high molecular weight DNA during centrifugal ultrafiltration in Microcon or Centricon units. Methods DNA Ladder As a model system to check for the introduction of single-strand nicks during the ultrafiltration spin, supercoiled Ladder DNA (Gibco-BRL, Gaithersburg, MD) which ranges in size from 2 to 8 kb was used. DNA diluted with TE buffer was loaded into Microcon 30K NMWL and Centricon 30K NMWL devices. The Microcon units were spun for 7 minutes at 12,000 x g, and the Centricon units were spun for 30 minutes at 5,000 x g. The concentrated DNA sample (retentate) was diluted again with TE buffer (to 500 µl or 2 ml, depending on the device used) and spun once more, as described above. The dilution step was repeated a third time. After the third concentration spin, the retentate was collected by placing the sample reservoirs upside down in new vials and spinning the units for 1 minute at 1,000 x g. The concentrated DNA was run on a 0.9% SeaKem GTG agarose gel (FMC) and stained with ethidium bromide to monitor sample integrity (Figure 17.1). DNA bands in lane 1 (starting material) are indistinguishable from the bands on lanes 2 4, which were spun repeatedly in the ultrafiltration devices. There is no evidence that any supercoiled DNA was converted to the relaxed or linear forms during the concentration procedure, which would be the case had single-strand nicks been introduced during centrifugation. 48

51 Discrete Size Plasmids The next set of experiments monitored the effect of centrifugal ultrafiltration on single population, discrete size plasmids. The plasmids used were pbr322 (4,361 bp; New England Biolabs, Inc.), pspt18 (3,104 bp; Boehringer Mannheim) and pxtl (10,400 bp; Stratagene). Samples (1 µg) of each plasmid were spun in Microcon 30K NMWL or Centricon 30K NMWL units, as described previously. The starting material and retentates were run on a 1% agarose gel and stained with ethidium bromide. The results are shown in Figure As in the case of the DNA Ladder, the concentrated samples (lanes 2, 3, 5, 6, 8, and 9) appear to be identical with their corresponding starting material. Again, there is no evidence of conversion of the supercoiled form to the relaxed form after exposure to g forces of 5,000 x g for 90 minutes and 12,000 x g for 21 minutes during the concentration spins. Genomic Size DNA To monitor the integrity of molecules in the size range of genomic DNA after centrifugation, lambda DNA (49 kb; Boehringer Mannheim, Indianapolis, IN) and Bsu36 l digested BacPAK6 DNA (125 kb) was used. The samples were diluted and centrifuged as described above. The retentates and starting material were run on a 1% agarose gel in a CHEF-DR II pulsed field electrophoresis system (Bio-Rad, Richmond, CA). Electrophoresis was performed at 200 V at 14 C in 0.5X TBE with ramped pulse from 1 to 6 seconds over 14 hours. The results with the lambda DNA mimic those of the other samples run in these sets of experiments. No adverse effects are noted after spinning the DNA at g forces up to 12,000 x g in the ultrafiltration units. However, the larger BacPAK 6 DNA does show some degradation after ultrafiltration at both 5,000 and 12,000 x g (Figure 17.3, smearing in lanes 5 and 6). Although a large percentage of the sample appears to be intact, there was loss of integrity of the BacPAK6 DNA sample after the concentration procedure. Conclusions DNA samples of up to 49 kb were concentrated repeatedly without any loss of sample integrity. Some loss of integrity was observed with a 125 kb sample, although it was not complete and represents a small percentage of the total DNA in the sample. For large fragments of DNA, centrifugal ultrafiltration provides a fast and efficient method to concentrate or desalt the sample. It results in high recovery of intact product. References 1. Takagi S, Kimura M, Katsuki M. BioTechniques 1993;14(2): PCR is covered by U.S. patents issued to Hoffmann-LaRoche, Inc. 3. Sheng N, Zhang J, Whitton JL, McKee T. BioTechniques 1993;14(5): Ruano G, Pagliaro EM, Schwartz TR, Lamy K, Messina D, Gaensslen RE, Lee HC. BioTechniques 1992;13(2): Figure 17.2 Specific plasmid DNA Lane 1: pbr322 starting material Lane 2: pbr322 spun in Microcon 30K NMWL device Lane 3: pbr322 spun in Centricon 30K NMWL device Lane 4: pspt18 starting material Lane 5: pspt18 spun in Microcon 30K NMWL device Lane 6: pspt18 spun in Centricon 30K NMWL device Lane 7: pxt1 starting material Lane 8: pxt1 spun in Microcon 30K NMWL device Lane 9: pxt1 spun in Centricon 30K NMWL device Figure 17.3 Large fragment DNA Lane 1: Lambda DNA starting material Lane 2: Lambda DNA spun in Microcon 30K NMWL device Lane 3: Lambda DNA spun in Centricon 30K NMWL device Lane 4: BacPAK DNA starting material Lane 5: BacPAK DNA spun in Microcon 30K NMWL device Lane 6: BacPAK DNA spun in Centricon 30K NMWL device 49

52 Protocols Nucleic Acids DNA Extraction from Agarose Gels with Montage Gel Extraction Kit or Ultrafree-DA Centrifugal Filters Introduction The Ultrafree-DA device is designed to recover 100 to 10,000 bp DNA from agarose gel slices in one 10-minute spin. It consists of a pre-assembled sample filter cup with an agarose gel nebulizer and a microcentrifuge vial. The device uses gel compression to extract DNA from the agarose. Centrifugal force collapses the gel structure, drives the agarose through a small orifice in the gel nebulizer and captures the resultant gel slurry in the sample filter cup. As the agarose is compressed at 5,000 x g, DNA is extruded from the gel's pores. The gel matrix is retained by the microporous membrane, and the DNA passes freely through the membrane. DNA can then be recovered in the filtrate vial. The Montage Gel Extraction Kit consists of 50 Ultrafree-DA centrifugal filters as well as a modified TAE buffer that allows the casting and running of the gel from which the DNA fragment is to be extracted. DNA prepared with the Ultrafree-DA centrifugal filter requires no further purification for most applications, including cloning and radioisotopic or fluorescent DNA sequencing. Since agarose gel electrophoresis has high resolving power, the small and large non-specific amplification products that frequently interfere with cloning and sequencing after PCR (polymerase chain reaction) are completely removed from the product. Materials Microcentrifuge Pre-assembled Ultrafree-DA centrifugal filter device or Montage Gel Extraction Kit Modified TAE* electrophoresis buffer (40 mm Tris-acetate, ph 8.0, 0.1 mm Na 2 EDTA) SeaKem agarose (FMC BioProducts) or equivalent Long-wavelength UV lamp Scalpel or razor blade Procedure 1. Electrophorese 30 µl of PCR product or other DNA through a <1.25% ordinary agarose gel, prepared in modified TAE buffer with ethidium bromide (0.5 µg/ml). 2. Locate the band of interest with a long wavelength UV lamp or transilluminator. With a razor blade or scalpel, cut out the slice of agarose (<100 µl or 100 mg) containing the band of interest. Trim any excess agarose away from band. 3. Place the gel slice into the gel nebulizer/sample filter cup/filtrate vial assembly and seal the device with the cap attached to vial. *Modified TAE is recommended rather than TBE for the following reasons: (1) TBE buffer strongly inhibits DNA sequencing reactions while modified TAE buffer does not. (2) Modified TAE has 0.1 mm Na 2 EDTA while regular TAE has 1.0 mm Na 2 EDTA. The EDTA level at 0.1 mm Na 2 EDTA will not interfere with the magnesium concentration in sequencing reactions and other downstream enzymatic treatments, many of which are dependent on magnesium. 50

53 4. Spin at 5,000 x g for 10 minutes. Centrifugation forces the agarose through the gel nebulizer, converting it to a fine slurry that is captured by the sample filter cup. Extruded DNA in electrophoresis buffer passes through the microporous membrane in the sample filter cup and collects in the filtrate vial. 5. DNA in the filtrate is now ready for sequencing or cloning without further purification. Discard the gel nebulizer and sample filter cup and store the DNA in the capped filtrate vial. Results Gel compression is a quick and easy technique for recovering DNA from an agarose gel slice. Table 18.1 Effect of gel disruption on typical DNA recoveries from agarose gels % DNA Recovered % DNA Recovered from Gel Disrupted DNA Size (bp) from Intact Gel by Gel Nebulizer ND * * * 29 * = Not detectable 51

54 Protocols Nucleic Acids RNA Purification with Microcon or Centricon Centrifugal Filters Introduction Figure 19.1 Concentration of RNA transcript. RNA is intact and recovered with high efficiency. Lane 1: Starting material Lane 2: RNA concentrated in a Microcon 100K NMWL device Lane 3: RNA concentrated in a Microcon 30K NMWL device When working with RNA, introduction of RNase contamination during sample preparation is of major concern. Sample yield and integrity can impact the efficiency of subsequent translations, hybridizations, protein binding, antisense, or ribozyme studies. A series of experiments was performed to determine the effectiveness of using Amicon centrifugal ultrafiltration devices from Millipore to concentrate and diafilter RNA samples. The results indicate remarkably high RNA recoveries and low adsorption losses especially with prior membrane passivation Table 19.1 TCA and total counts of unpurified and purified RNA samples Unpurified Centricon-purified Transcript Transcript TCA counts 70,000 68,500 Total counts 136,500 69, kb 0.68 kb Methods RNA Sample Integrity The primary objective of the study was to determine the effect of centrifugation on RNA integrity. The second objective was to determine any gross changes in the RNA that could be a result of contact of the sample with the ultrafiltration devices themselves. An RNA transcript was made, using MEGAscript T7 RNA polymerase (Ambion) and DNA template Riboprobe Gemini (Promega) according to manufacturer s protocols. This transcript served as the starting RNA sample for the experiments discussed below. Figure 19.1 is an autoradiogram of labeled RNA. It compares an untreated sample to samples that were concentrated in Microcon 100K NMWL and 30K NMWL devices, used directly as supplied. The Microcon 100K NMWL unit was spun at a force of 3,000 x g and the Microcon 30K NMWL unit at 12,000 x g. As is evident from the autoradiogram, both unfiltered samples are virtually indistinguishable from the starting material. Similar results were obtained with Centricon units, spun at 1,000 x g (Centricon 100K NMWL unit) or 5,000 x g (Centricon 30K NMWL unit). Diafiltration of RNA Samples Experiments were run to determine the RNA recovery and diafiltration efficiency of Centricon devices. Centricon 100K NMWL units were used to remove unincorporated, radiolabeled ribonucleotides from the RNA transcript. Two ml of distilled water were placed into the Centricon sample reservoir, then 90 µl of the unpurified RNA transcript were added. The device was spun for 30 minutes at 1,000 x g, then inverted and spun for 2 minutes at 1,000 x g to collect the concentrated RNA (retentate). The retentate was diluted 1:10 with water. Total and TCA precipitable counts were measured. 52

55 If the Centricon device effectively removed the unincorporated ribonucleotides, Total and TCA counts should be close in value. TCA counts of the retentate sample and of the unpurified transcript (starting material) should also be similar if ultrafiltration resulted in high recovery of the transcript. Average values (n=3) of the Total and TCA counts appear in Table For the retentate sample, the Total and TCA counts are similar, indicating >95% removal of the nucleotides. TCA counts of the samples indicate that RNA recovery was also above 95%. RNA Recovery RNA recovery is a function of the initial RNA concentration and the buffer salt concentration. Figure 19.2 shows RNA transcript recovery in Microcon 100K NMWL and Centricon 100K NMWL units as a function of RNA concentration. For all concentrations evaluated, retentate recovery is above 85%, even at an initial concentration as low as 25 ng/ml. Figure 19.2 also displays the material monitored in the filtrate and on the membranes of the Microcon units. (Membranes were removed and counted without further treatment.) The amount of RNA in the filtrate does not vary significantly as a function of concentration. The RNA recovered on the membranes varies from <1% at high initial concentrations to 4% at the lowest concentration tested. Millipore s Amicon centrifugal ultrafiltration devices contain low-binding cellulosic Ultracel-YM membranes. Nitrocellulose is commonly used to immobilize RNA, generally in high salt concentrations. Figure 19.3 shows RNA recovery as a function of buffer salt concentration. The data show that both retentate and total recoveries fall as salt concentration increases. An increase in the amount of material on the membrane is also seen (from 2% to 6%) as the salt concentration increases from 10 to 500 mm. Similar trends are seen in the use of Microcon 100K NMWL devices, except that retentate recoveries fall to 60% at 500 mm salt. It was possible to count the devices themselves in addition to the filtrate and membrane. The results show a slight increase in counts on the membrane with increasing salt concentration. Significantly more (from 9% to 17%) material remained on the device as salt concentration increased. RNA Recovered 100% 80% 60% 40% 20% 0% RNA Concentration (µg/ml) Microcon Filtrate Microcon Membrane Microcon Retentate Centricon Retentate Figure 19.2 RNA recovery in Centricon and Microcon concentrators. RNA was concentrated as described in text. Cherenkov counts of Centricon retentate and Microcon retentate, filtrate and membrane (n=3 units) were compared to total starting counts. RNA Recovered 100% 80% 60% 40% 20% 0% NaCl Concentration (mm) Filtrate Membrane Device Retentate Figure 19.3 Effect of increasing buffer salt concentration on RNA recovery with Centricon devices (two left bars at each concentration) or Microcon devices (two right bars at each concentration). Radiolabeled RNA samples were spun at 1,000 x g for 30 minutes in a Centricon device or at 3,000 x g for 15 minutes in a Microcon device. Retentate was collected by inverting the unit and spinning for 2 minutes (Centricon devices) or 1 minute (Microcon devices) at 1,000 x g. Cherenkov counts of retentate, filtrate, membrane, and device (Microcon units only) were compared to total starting counts. 53

56 RNA Recovered 100% 80% 60% 40% 20% 0% NaCl Concentration (mm) Filtrate Membrane Device Retentate Figure 19.4 Improvement in RNA recoveries using Microcon devices (n=6) which were pre-treated with a 5% SDS solution. Samples were run as described in Figure Passivation increased RNA recovery to 80%, regardless of initial salt concentration (Figure 19.4). This is attributed to a decrease in the amount of RNA adhering to the device. When the devices were passivated, RNA loss due to adsorption was reduced to 2%, regardless of salt concentration. Conclusion The results shown here demonstrate that RNA samples can be concentrated or diafiltered with Centricon and Microcon concentrators without loss of RNA integrity. RNA recovery in the retentate is typically >85%. As the salt concentration of the buffer system increases, RNA retentate recovery decreases, principally due to RNA adhering to the device rather than to the membrane. RNA recovery from solutions with high initial salt concentrations can be significantly improved by pre-treating the devices with a 5% SDS solution. 54

57 Protocols Nucleic Acids Enzyme Removal with Micropure-EZ Centrifugal Filters Introduction The Micropure-EZ device is a convenient device for removing enzymes from double-stranded (ds) DNA (20 bp to >50,000 bp) solutions in a single centrifugation. It can be used to remove enzymes whenever heat inactivation or phenol/chloroform extraction is impossible or impractical. After an enzyme reaction, the modified DNA solution containing dilute protein is simply pipetted into a Micropure-EZ device that has been inserted into a vial (which is supplied) and centrifuged for 1 minute at 12,000 14,000 x g. The DNA passes freely into the vial (typically with 85 90% DNA recovery) while the enzyme remains bound to the proprietary membrane in the Micropure-EZ device. Removal is defined as the complete absence of detectable enzyme activity or, as in the isolated case of T4 polynucleotide kinase, inconsequential residual activity (<0.08%). For simultaneous concentration of the enzyme-free DNA, place the Micropure-EZ device into a Microcon microconcentrator (Figure 20.1) before centrifuging again. The purified DNA is suitable for cloning or for other enzymatic manipulation. While most enzymes were removed by the Micropure-EZ device (Table 20.1, on page 57), Millipore felt it was equally important to notify customers of several enzymes that were not removed (Table 20.2). Millipore scientists were very careful to rule out enzyme inhibition as a mechanism. The sensitivity of each assay to detect trace amounts of restriction enzymes in Micropure-EZ filtrates was determined by carrying out a dilution series (this is the method used by restriction enzyme manufacturers to estimate total activity). Figure 20.1 Operation of Micropure-EZ device Method DNA Micropure-EZ Vial Enzyme 30 seconds Salt Remove Enzyme Restriction and other enyzmes are absorbed by Micropure-EZ. dsdna passes freely. Micropure-EZ Microcon Assembly In order to ensure that each enzyme assay was detecting trace amounts of restriction enzymes in Micropure-EZ filtrates, serial dilutions of Bgl I (representing 2 units, 0.4 units, 0.08 units, and units of total activity in 10 µl) were carried out in a reaction mix* prepared with either sterile DI water (sdh 2 O) or with sdh 2 O that had been filtered through Micropure-EZ and then incubated. Comparison of the resultant restriction digests after agarose gel electrophoreses verified that aqueous extractables from Micropure-EZ devices did not inhibit the enzyme. Also, these standard curves determined the sensitivity of the enzyme assay (i.e., the theoretical amount of residual enzyme activity that could be detected, if present). 3 minutes Remove Enzyme Restriction and other enyzmes are absorbed by Micropure-EZ. dsdna passes freely. Concentrate DNA DNA is retained by ultrafilter in Microcon. Salts pass freely. Recover 85% DNA *15.8 µl of sdh 2 O, 2 µl of 10X NEBuffer 2, 0.2 µl of 100X BSA, and 2 µl of Bgl I (10 units/µl) 55

58 To stress test Micropure-EZ devices sufficiently with enzymes and DNA, several extreme operating conditions were used consistently. Micropure-EZ devices were challenged with 50 units of Bgl I in the presence of decoy DNA (1 µg of pbr322) and 5 µg of bovine serum albumin (BSA). Devices were spun at 14,000 x g for 30 seconds. The filtrates were then assayed for residual enzyme activity by adding 1 µl of puc19 DNA and 0.5 µl of 100X BSA to the filtrate, mixing thoroughly and incubating at 37 C for 1 hour. After a brief centrifugation to collect the condensate at the bottom of the microcentrifuge tubes, 1 µl of 0.5 M disodium EDTA and 10 µl of 5X loading buffer were added to stop the reaction. The negative control was a portion of DNA master mix. Control digests of pbr322 DNA and puc19 DNA were carried out separately. Results and Discussion The Bgl I standard curve made with the Micropure- EZ-filtered sdh 2 O was indistinguishable from the standard curve made with untreated sdh 2 O. The plasmid DNA (1 µg of puc19 and 1 µg of pbr322) was cut to completion by 2 units of Bgl I after one hour at 37 C (Figure 20.2, lanes 1 and 6). None of the enzymes shown in Tables 20.1 or 20.2 was measurably inhibited by Micropure-EZ-filtered sdh 2 O. The standard curves indicated that as little as 0.08 units of Bgl I could be detected by this assay (lanes 3 and 8). The devices removed the 50 units of Bgl I as evidenced by an absence of detectable activity in the filtrates. In the lanes corresponding to the Bgl I challenged Micropure-EZ inserts, some activity was observed against the decoy pbr322 DNA, as expected during its brief exposure to Bgl I (particularly evident in lane 14). However, the puc19, which was added directly to the filtrate and incubated, appeared completely intact (lanes 11 14). Since the standard curve indicated that 0.08 units would be detected if it were present, we calculated that at least 99.8% of Bgl I was removed and/or inactivated. Removal, rather than inactivation, is the probable mechanism by which Micropure-EZ devices operate, since in separate experiments we were unable to detect any enzyme (irrespective of activity) in Micropure-EZ filtrates using very sensitive HPLC methods (data not shown). Summary Enzyme removal and excellent DNA recovery are accomplished with Micropure-EZ devices in a single 60-second spin without any pre-wetting, binding, washing or elution step. Cumbersome phenol/ chloroform extraction and the associated hazardous waste accumulation are avoided. An additional line of evidence for the suitability of Micropure-EZ devices in molecular biology applications is that restriction digest purified with Micropure-EZ devices alone are readily ligated and cloned into plasmid DNA (M.H.A.L., unpublished observations). Micropure-EZ devices used alone or with Microcon devices permit rapid and efficient sequential enzymatic DNA manipulations. Figure 20.2 Analytical agarose gel, demonstrating the detection limits for Bgl I and Bgl I removal by Micropure-EZ devices. Lanes 1 4: Serial dilutions of BgI I representing 2 units, 0.4 units, 0.08 units, and units of activity (respectively) against the DNA master mix prepared in non-filtered sdh 2 O Lane 5: Undigested DNA master mix used as substrate for the standard curve containing puc19 and pbr322 DNA Lanes 6 9: Inhibition control: serial dilutions of BgI I representing 2 units, 0.,4 units, 0.08 units, and units of activity (respectively) against the DNA master mix prepared in Micropure-EZ-filtered sdh 2 O Lane 10: Molecular weight standard: 1 kb DNA ladder (Gibco-BRL, Gaithersburg, MD) Lanes 11 14: Filtrates incubated with puc19 DNA after challenging Micropure-EZ devices with 50 units of BgI I, BSA and decoy pbr322 DNA Lane 15: Uncut pbr322 Lane 16: BgI I cut pbr322 Lane 17: Uncut puc19 Lane 18: BgI I cut puc

59 Table 20.1 Enzymes removed by Micropure-EZ devices In tests, all enzymes were removed from solutions containing DNA or RNA (in the case of RNase A). Five µg of bovine serum albumin were also present during removal of restriction enzymes. Removal was indicated by undetectable or inconsequential enzyme activity in filtrate (< 0.08% residual in the case of T4 polynucleotide kinase). Enzyme Challenge AMV reverse transcriptase 50 U Calf intestinal alkaline 10 U phosphatase DNase I (bovine pancreas) 10 U Exonuclease III (E. coli) 100 U MMLV reverse transcriptase 600 U Mung bean nuclease 50 U Proteinase K (Amresco) 5 µg T4 DNA ligase 2,000 U* T4 DNA polymerase 15 U T4 polynucleotide kinase** 50 U Taq DNA polymerase 5 U Terminal deoxynucleotidyl 45 U transferase Acc I 50 U Enzyme Apa I BamH I Bcl I Bgl I BsiW I BssH II BstN I Dpn I EcoR I Hae III Hinc II Hind III Hpa I Kpn I Mbo I Challenge 100 U 100 U 50 U 50 U 70 U 20 U 50 U 100 U 100 U 100 U 50 U 100 U 25 U 50 U 25 U Enzyme Mlu I Nco I Nde I NgoM I Nhe I Not I Nru I Pst I Sac I Sac II Sal I Sca I Sph I Sst I Xho I Challenge 50 U 50 U 100 U 50 U 25 U 50 U 50 U 100 U 100 U 100 U 100 U 50 U 25 U 50 U 100 U *New England Biolabs unit definition **T4 polynucelotide kinase is not recommended with oligonucleotides (dsdna only). The results suggest that this kinase mediates the binding of oligos to the membrane in Micropure-EZ device, causing oligo loss. Table 20.2 Enzymes not removed by Micropure-EZ devices In tests, Micropure-EZ did not remove the indicated number of units of the listed enzymes. It may be effective in removing a lower number of units. Enzyme Challenge Enzyme Challenge Enzyme Challenge Bacterial alkaline phosphatase 0.6 U ApaL I 10 U Msp I 100 U DNA polymerase I (Klenow) 20 U Bgl II 50 U Pvu I 25 U Exonuclease I 50 U BsoB I 50 U Pvu II 50 U Pfu DNA polymerase 2.5 U Cla I 25 U Sau3A I 20 U Vent DNA polymerase 4 U Eae I 15 U Sfi I 50 U Shrimp alkaline phosphatase 1 U EcoR V 50 U Sma I 25 U RNase A (bovine pancreas) 1 µg Hinf I 50 U Xba I 50 U T4 RNA ligase is not recommended. Results suggest this ligase mediates binding of nucleic acids to the membrane in Micropure-EZ causing sample loss. 57

60 Protocols High Throughput High Throughput Applications Figure 1. MultiScreen Filter Plate with Ultracel-10 ultrafiltration membrane The MultiScreen filter plate with Ultracel-10 membrane provides a new method for high throughput sample prep. The ultrafiltration-based filter plate is designed for use with centrifugation and is compatible with standard microtiter plates, instrumentation, and liquid handling equipment. All of the publications summarized below can be supplied by your local Millipore office or downloaded from Nucleic Acid Purification and Concentration AN1040EN00 The MultiScreen Filter Plate with Ultracel-10 membrane can be used to purify and concentrate single-stranded oligonucleotides and doublestranded DNA fragments, as well as plasmid and genomic DNA. Concentration of Proteins in Cell Lysate AN1424EN00 The MultiScreen filter plate with Ultracel-10 membrane can be used to concentrate whole cell lysates without loss of protein and with high reproducibility across the plate. Applications include parallel protein purification, protein concentration and buffer exchange in cell lysates for subsequent separation or assaying. Purification of Serum Peptides for Biomarkers Research AN2010EN00 This study is a high throughput version of the application nate on page 24 of the Protocols section of this handbook. Enzymatic Activity Recovery AN2011EN00 This study demonstrates the use of the MultiScreen plate with Ultracel-10 membrane for concentrating alkaline phosphatase without loss of biological activity and with high reproducibility across the plate. 58

61 Glossary Asymmetric Membrane 1 Membrane constituted of two or more structural planes of non-identical morphologies. Batch Process A fixed volume of solution contained in a tank to which the concentrate is returned during the process. Composite Membrane 1 Membrane having chemically or structurally distinct layers. Concentration Polarization Accumulation of rejected solute on the membrane surface. Depends on interactions of pressure, viscosity, crossflow (tangential) velocity, fluid flow conditions, flow channel conditions and temperatures. Crossflow (Tangential Flow) Solution flows across (tangential to) a membrane surface. Facilitates back diffusion of solute from that surface into the bulk solution, counteracting concentration polarization. Diafiltration Removal of small components from the retained species during ultrafiltration by adding water or buffer solution to the retentate. See page 7 for further discussion. Dialysis Diffusive transport of ions or other small molecules through a membrane barrier that contacts solvent on both sides. Downstream 1 Side of a membrane from which the permeate emerges. Feed (Sample) The starting solution (sometimes the solution remaining upstream of the membrane). Fluid Velocity The flow rate of solution across the membrane surface in cross (tangential) flow. Related to hydraulic pressure drop. Flux The filtration rate through the membrane per unit area. Fouling Irreversible decline in membrane flux due to deposition and accumulation of submicron particles and solutes on the membrane surface. Also, crystallization and precipitation of small solutes on the surface and in the pores of the membrane. Not to be confused with concentration polarization. Membrane 1 Structure having lateral dimensions much greater than its thickness, through which mass transfer may occur under a variety of driving forces. Molecular Weight Cut-off (MWCO) See Nominal Molecular Weight Limit. Nanofiltration 1 Pressure-driven membrane-based separation process in which particles and dissolved molecules smaller than about 2 nm are rejected. Nucleotide Cut-off (NCO) The number of nucleotides in a DNA fragment (single- or double-stranded) at which 90% of the fragment is retained by the membrane. Nominal Molecular Weight Limit (NMWL) The molecular weight at which at least 90% of a globular solute of that MW is retained by the membrane. Permeate (Filtrate, Ultrafiltrate) The solution passing through the membrane, containing solvent and solutes not retained by the membrane. Plugging Accumulation of debris on the fluid flow path, restricting or blocking flow. Rejection The fraction of solute held back by the membrane. Can be measured at any point in the process or averaged over the run. Retentate (Reject Stream, Concentrate) The solution containing the retained (rejected) species. Retention Factor 1 (r F ) Parameter defined as one minus the ratio of permeate concentration to the retentate concentration of a component. Note: r F = 1 [p]/[r] where [p] = concentration of solute in permeate, and [r] = concentration of solute in retentate. Reverse Osmosis 1 Liquid-phase pressure-driven separation process in which applied transmembrane pressure causes selective movement of solvent against its osmotic pressure difference. Tangential Flow Filtration (TFF) Flow through a membrane device in which the fluid on the upstream side moves parallel to the membrane surface. Transmembrane Pressure (TMP) The driving force in ultrafiltration. In a stirred cell, equivalent to gas pressure. In centrifugal devices, it is related to g-force. In a flowing system, TMP decreases as the stream moves from inlet to outlet. Average TMP = [(P in + P out )/2] P permeate Ultrafiltration 1 Pressure-driven membrane-based separation process in which particles and dissolved molecules smaller than 0.1 µm and larger than about 2 nm are rejected. Yield Amount of species recovered at the end of the process as a percentage of the amount present in the feed solution. References 1. Terminology for membranes and membrane processes (IUPAC Recommendations 1996). IUPAC, Journal of Membrane Science. 1996;120(1):

62 Acknowledgements Millipore wishes to acknowledge the efforts of the following employees in developing this reference book: Elena Chernokalskaya, Ph.D. Manager, Proteomics Applications Mark Kavonian Worldwide Group Product Manager Protein Research Products Richard Leary Technical Service Specialist Jack T. Leonard, Ph.D. R&D Director, Life Science Research Applications Peter Rapiejko Millipore also wishes to acknowledge the following individuals for their contributions: Eduardo Vottero, University of British Columbia Peter A. Lemaire and Dr. James Cole, University of Connecticut Gary Smejkal, Proteome Systems, Woburn, MA Leonid Kryazhev, Genome Quebec, Montreal, Canada Mathew L. Thakur, Thomas Jefferson University Hospital, Philadelphia, PA Mark Merchant, Helena Laboratories, Beaumont, TX Department of Forensic Biology, New York City Patrick O. Brown, Max Diehn, Ash Alizadeh, Stanford University School of Medicine Dr. Sophia N. Karagiannis, GKT School of Biomedical Sciences, London 60

63 Patent PCR is covered by US patents issued to Hoffmann-LaRoche, Inc. Trademarks Millipore, Amicon, Biomax, Centricon, Centrilutor, Centriplus, Centriprep, Durapore, Immobilon, Microcon, Micropure, Milli-Q, Minicon, Montage, MultiScreen, Pellicon, Prep/Scale, PROSEP, Ultracel, Ultrafree, Zip, and ZipTip are trademarks of Millipore Corporation. ProteoPrep is a trademark of Sigma-Aldrich Biotechnology LP. ProteomIQ, ElectrophoretIQ, IsoelectrIQ, and GelChips are trademarks of Proteome Systems. SpectraFLUOR is a trademark of Tecan Group AG. DryStrips, Sephadex, Sephacryl, Sepharose, and Storm are trademarks of Amersham Biosciences AB. CHEF-DR II and Chelex are trademarks of Bio-Rad Laboratories. MEGAscript is a trademark of Ambion Inc. GenePrint, Flexi and Riboprobe are trademarks of Promega Corporation. SeaKem is a trademark of FMC. AmpFLSTR and Voyager-DE are trademarks of Applera Corporation or a subsidiary. Triton is a trademark of Union Carbide Chemicals & Plastics Technology Corporation. Biofuge is a trademark of Kendro Laboratory Products, Gmbh. NuPage, SimplyBlue, Superscript and Xcell Surelock are trademarks of Invitrogen Corporation. Qiagen is a trademark of Qiagen Gmbh. Speed Vac is a trademark of Thermo Savant. BacPAK and Talon are trademarks of Becton, Dickinson and Company. Vent is a trademark of New England Biolabs, Inc. Coomassie is a trademark of Imperial Industries, PLC. Tween is a trademark of Atlas Powder Company.

64 Additional Tools for Life Scientists From Amicon centrifugal devices to Mill-Q water systems to Zip micro-spe technology, no one offers a broader range of sample preparation tools than Millipore. Â Blotting Proteins Immobilon are the most widely used PVDF membranes for western, dot/slot, and other protein blots. Request a free copy of our 56-page Protein Blotting Handbook at Nucleic acids Immobilon-Ny+ are positively charged nylon membranes that provide reliable transfer, hybridization, and reprobing with maximum sensitivity and minimal background for Southern blotting, northern blotting, and colony/plaque lifts. Sterile Filtration and Chromatography Sample Prep Membranes and devices for sterile and non-sterile filtration Millipore offers hundreds of membrane-based devices for sterile filtration, chromatography sample prep, and almost any other application in the life sciences laboratory. DNA Sequencing Kits and devices for nucleic acid sample prep Montage life science kits use Millipore s size-exclusion technology for high quality plasmid mini-preps and PCR and sequencing reaction cleanup. Laboratory Water Systems Pure water solutions Millipore provides total solutions from bench-top systems to custom-engineered purification chains for laboratory buildings. You ll find Millipore water systems installed in over 70,000 laboratories worldwide supplying pure water for electrophoresis, PCR, chromatography, and other life science applications. Bookmarks The life sciences change every day. And so does millipore.com. We re continually adding new content and new functionality. Millipore life sciences home page (on the banner, under Applications ) On-line ordering Order tracking Technical Support Millipore Technical Service Specialists support ultrafiltration and many other life science applications, including blotting, DNA sequencing sample prep, sterile filtration, and MS sample prep. To contact a Specialist, call your local office or submit a question at techservice. To access our library of frequently asked questions, go to Lit. No. TP0040EN00 Rev. Printed in U.S.A / Millipore Corporation, Billerica, MA U.S.A. All rights reserved.