Products for Electrophoresis and Staining. Gel Electrophoresis of Proteins

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1 Products for Electrophoresis and Staining 2 Gel electrophoresis is a technique in which charged molecules, such as protein or DNA, are separated according to physical properties as y are forced through a gel by an electrical current. Proteins are commonly separated using polyacrylamide gel electrophoresis (PAGE) to characterize individual proteins in a complex sample or to examine multiple proteins within a single sample. PAGE can be used as a preparative tool to obtain a pure protein sample, or as an analytical tool to provide information on mass, charge, purity or presence of a protein. Several forms of PAGE exist and can provide different types of information about protein(s). Nondenaturing PAGE, also called native PAGE, separates proteins according to ir mass:charge ratio SDS-PAGE, most widely used electrophoresis technique, separates proteins primarily by mass Two-dimensional PAGE (2-D PAGE) separates proteins by isoelectric point in first dimension and by mass in second dimension Contents Gel Preparation Homemade Gel Recipes...3 Precast Gels...4 Isoelectric Focusing and 2-D Gels...5 Native Page...5 SDS Page...6 Reagents for Gel Preparation and Precast Gels Sample Preparation For SDS Page...9 For 2-D Gels...11 Products for Sample Preparation...12 Buffer Preparation Sample and Running Buffer Recipes Products for Buffer Preparation Molecular Weight Markers Molecular Weight Marker Products...17 Gel Staining General In-Gel Detection of Protein Bands...21 Specific Functional Group Stains...22 Gel Staining Products...24 Western Blotting Visit our web site ( for information on hazard codes listed in ordering information boxes.

2 Electrophoresis & Staining Flowchart 1. sample Buffers Choose MW markers Run Stain Post staining Step 1 Homemade Gel Recipes Acrylamide is material of choice for preparing electrophoretic gels to separate proteins by size. Acrylamide mixed with bisacrylamide forms a cross-linked polymer network when polymerizing agent, ammonium persulfate, is added (Figure 1). The ammonium persulfate produces free radicals faster in presence of TEMED (N,N,N,N -tetramethylenediamine). The size of pores created in is inversely related to amount of acrylamide used. For example, a 7% polyacrylamide gel will have larger pores in than a 12% polyacrylamide gel. Gels with a low percentage of acrylamide are typically used to resolve large proteins and gels with a high percentage of acrylamide are used to resolve small proteins. Table 1 provides recipes for preparing gels with different acrylamide concentrations. Pierce offers many of raw materials necessary for preparing PAGE gels, all of which are supplied with minimal contaminants. For example, Pierce SDS (Product # 28312) is a high-grade material, containing at least 98% of C 12 alkyl sulfate chain length, with minimal presence of C 14 or C 16 chain length. This results in more consistent SDS-PAGE separations and improved renaturation of proteins for in situ enzyme activity. 1 Analysis of multiple samples is accomplished using a one-dimensional slab gel. Slab gel sizes commonly range from 15 cm x 18 cm down to 2 cm x 3 cm. Small gels typically require less time and reagents than ir larger counterparts and are suited for rapid screening. However, larger gels provide better resolution and are needed for separating similar proteins or a large number of proteins. Samples are applied at top of slab gel in sample wells that span width of. When electrical current is applied, proteins migrate down through matrix, creating lanes of protein bands. In native PAGE, migration occurs because most proteins carry a net negative charge at slightly basic ph. The higher negative charge density (more charges per molecule mass), faster a protein will migrate. At same time, frictional force of matrix creates a sieving effect, retarding movement of proteins according to ir size. Small proteins face only a small frictional force while large proteins face a larger frictional force. Thus native PAGE separates proteins based upon both ir charge and mass. In SDS-PAGE, proteins are treated with sodium dodecyl sulfate (SDS) before electrophoresis so that charge density of all proteins is made roughly equal. When se samples are electrophoresed, proteins are separated according to mass. SDS-PAGE allows estimation of molecular weight of proteins. In this application, a sample of unknown molecular weight is compared directly with proteins of known molecular weight (MW standards) in an adjacent lane. SDS-PAGE is also used for routine separation and analysis of proteins because of its speed, simplicity and resolving capability. Step 1 CH 2 CH C O NH 2 Acrylamide + CH 2 CH C O NH CH 2 NH C O CH 2 CH BIS Bis CH 2 CH CH 2 CH CH 2 CH C O C O C O NH 2 NH 2 NH CH 2 NH NH 2 NH 2 C O C O C O CH 2 CH CH 2 CH CH 2 CH H Polyacrylamide Persulfate TEMED Figure 1. Polymerization and cross-linking of acrylamide. Step 1 3 Tel: or

3 Step 1 4 Table 1. SDS-PAGE Formulas for Mini-Gels (8.0 cm x 8.0 cm) Running Gel Percent Acrylamide Gel 7% 10% 11% 12.5% 40% Acrylamide Solution (w/v) 5.25 ml 7.5 ml 8.25 ml ml 1% Bisacrylamide 4.8 ml 3.9 ml 3.6 ml 3.1 ml 1.5 M Tris HCI, ph ml 7.5 ml 7.5 ml 7.5 ml ********** Add distilled water to bring total volume to 30 ml ********** 10% Ammonium Persulfate (Product # 17874) 0.3 ml 0.3 ml 0.3 ml 0.3 ml 10% SDS, C12 grade (Product # 28312) 0.3 ml 0.3 ml 0.3 ml 0.3 ml TEMED (Product # 17919) 0.03 ml 0.03 ml 0.03 ml 0.03 ml Stacking Gel 7% Acrylamide Gel 40% Acrylamide Solution (w/v) 0.75 ml 1% Bisacrylamide 0.1 ml 0.5 M Tris HCI, ph ml Deionized Water 5.6 ml 10% Ammonium Persulfate (Product # 17874) 0.1 ml 10% SDS, C12 grade (Product # 28312) 0.1 ml TEMED (Product # 17919) 0.01 ml Running Buffer: 25 mm Tris, 192 mm Glycine and 0.1% SDS, ph 8.3 Use: BupH Tris-Glycine-SDS buffer (Product # 28378) Sample Buffer: 0.3 M Tris HCl, ph 6.8, 5% SDS, 50% glycerol, bright pink tracking dye Use: ImmunoPure Lane Marker Non-Reducing Sample Buffer (Product # 39001) For reducing gels use: ImmunoPure Lane Marker Reducing Sample Buffer (Product # 39000) that contains 100 mm Dithiothreitol (Product # 20290) Add one volume of Product # or to four volumes of protein sample. Boil for 3-5 minutes n cool to room temperature before applying 15 µl-25 µl in sample well. Coomassie Stain: 0.125% Coomassie Brilliant Blue R-250 (Product # 20278) 50% Methanol 10% Acetic Acid Coomassie Destaining Solution: 50% Methanol + 10% Acetic Acid } Alternatively, stain directly with GelCode Blue Stain Reagent (Product # 24592) Multiple components of a single sample may be resolved most completely by 2-D PAGE. The first dimension separates proteins according to isoelectric point (pi) and second dimension separates by mass. 2-D PAGE provides highest resolution for protein analysis and is a key technique in proteomic research in which resolution of thousands of proteins on a single gel is necessary. To obtain optimal resolution of proteins, a stacking gel is poured over top of resolving gel. The stacking gel has a lower concentration of acrylamide (larger pore size), lower ph and a different ionic content. This allows proteins in a lane to be concentrated into a tight band before entering running or resolving gel and produces a gel with tighter or better separated protein bands. The resolving gel may consist of a constant acrylamide concentration or a gradient of acrylamide concentration (high percentage of acrylamide at bottom of and low percentage at top). A gradient gel is prepared by mixing two different concentrations of acrylamide solution to form a gradient with decreasing concentrations of acrylamide. As gradient forms, it is layered into a gel cassette. A gradient gel allows separation of a mixture of proteins with a greater molecular weight range than a gel with a fixed acrylamide concentration. If a sample contains proteins with large differences in molecular weights, n a gradient gel is recommended. A stacking gel is unnecessary when using a gradient gel because continually decreasing pore size performs this function. Precast Gels While many researchers continue to pour acrylamide gels on a routine basis, a growing number have adopted some form of precast gel. Purchasing precast gels saves considerable time, and gels are available in a variety of percentages including difficult-to-pour gradient gels that provide excellent resolution and separate proteins over widest range of molecular weights. Anor reason to use precast gels is reproducibility offered by long shelf life versions of such gels that are poured consistently and that continue to perform consistently over time. Under conditions normally used to pour polyacrylamide gels, hydrolysis occurs, resulting in formation of acrylic acid from polyacrylamide. This indicates that performance of gel changes with time and places severe limits on useful shelf life of. In addition, precast polyacrylamide gels obviate need to work with acrylamide monomer a known neurotoxin and suspected carcinogen. For more product information, or to download a product instruction booklet, visit

4 Electrophoresis & Staining Flowchart 1. sample Buffers Choose MW markers Run Stain Post staining Precise Protein Gels from Pierce are cast in a durable plastic cassette using a neutral ph buffer that inhibits hydrolysis of polyacrylamide and allows us to guarantee performance of gels for one year. They are compatible with standard mini-gel tanks so re is no need to purchase specialized equipment. The Tris-HEPES-SDS running buffer produces excellent resolution of protein bands and short run times of only 45 minutes. Precise Gels can be stained using common methods or transferred efficiently for Western blotting in just 90 minutes. Isoelectric Focusing and 2-D Gels An isoelectric focusing gel (IEF gel) can be used to separate proteins according to charge and to determine ph at which a protein has a net charge of zero. This ph is known as pi of protein and is a distinguishing characteristic of protein that provides useful information for purifying and handling protein. The pi of a protein is determined by number of acidic and basic residues in protein. At physiological ph, carboxyl groups of acidic residues are predominantly deprotonated and impart a negative charge. In contrast, amine groups of basic residues are protonated and carry a positive charge. By identifying pi value of a protein, buffer systems for largescale purification can be designed. For example, a protein with a pi of 5.6 will have a net charge of zero in a solution at ph 5.6. As ph of buffer system is increased, this protein (pi 5.6) takes on an overall negative charge because carboxyls and amines are both deprotonated. The protein can n be purified on a DEAE ion exchange column in a buffer at ph 8.0. The negative charges will cause protein to be retained on positively charged column. To perform IEF, a ph gradient is established in a tube or strip gel using a specially formulated buffer system or ampholyte mixture. Ampholytes are a mixture of amino acid polymers that have surface charges corresponding to different ph ranges and are available as immobilized ph gradient (IPG) strips for consistency and convenience. A protein sample is loaded onto and electrodes are attached (anode at acidic end of gradient and cathode at basic end of gradient). Proteins with a net positive charge on surface will migrate to cathode when an electrical current is applied. Negatively charged proteins will move toward anode. When protein in ph gradient reaches a zone in which net surface charge is zero, it will no longer move. At this point protein becomes focused and a band is formed in. Following isoelectric focusing, a protein mixture may be separated in a second dimension by SDS-PAGE. This technique, known as 2-D PAGE, is used to resolve complex protein mixtures into greatest number of individual protein spots. The IEF gel is equilibrated with SDS and laid across top of an SDS-PAGE gel. Current is applied and proteins migrate into where separation occurs according to mass. This two-dimensional separation of proteins according to pi and mass allows resolution of proteins that would not normally be separated by a one-dimensional method. Several thousand protein spots may be resolved on a single 2-D PAGE gel, making this technique suitable for proteomics analysis (Figure 2). IEF First dimension, tube gel or strip gel Figure 2. Schematic illustration of 2-D PAGE. Native PAGE SDS Slab Gel Second dimension, slab gel Electrophoresis of a protein in its native state relies upon intrinsic charge of protein and its mass. For a protein to migrate into toward anode, it must have an overall negative charge at ph of /buffer system. For this reason, native PAGE is commonly performed at a slightly basic ph where proteins with a neutral or acidic pi will have required net negative charge. Alternatively, process can be done in reverse by using an acidic ph to impart a positive charge to most proteins. This method requires reversing anode and cathode because positively charged proteins will migrate toward cathode. In addition to charge of a protein, or factors such as size and shape of a protein also influence its mobility in native PAGE. Because no denaturants are present in native PAGE, subunit interactions within a multimeric protein are generally retained and information may be gained about quaternary structure. In addition, many proteins have been shown to be enzymatically active following separation by native PAGE. Thus, it may be used for preparation of purified, active proteins. 2 Following electrophoresis, proteins may be recovered from a native gel by passive diffusion or electroelution. 3 To maintain integrity of proteins during electrophoresis, it is important to keep apparatus cool and minimize effects of denaturation and proteolysis. Extremes of ph should generally be avoided in native PAGE as y may lead to irreversible damage, such as denaturation or aggregation, to protein of interest. Step 1 5 Tel: or

5 Step 1 SDS-PAGE In SDS-PAGE applications, sample applied to slab gel has been treated with detergent sodium dodecyl sulfate (SDS). This ionic detergent denatures proteins in sample and binds tightly to uncoiled molecule. The SDS molecules mask intrinsic charge of protein and create a relatively uniform negative charge distribution caused by sulfate groups on SDS. When an electric current is applied, all proteins will migrate through toward anode, which is placed at bottom of. The SDS-PAGE gel separates proteins primarily according to size because SDS-coated proteins have a uniform charge:mass ratio. Proteins with less mass travel more quickly through than those with greater mass because of sieving effect of matrix. Protein molecular weights can be estimated by running standard proteins of known molecular weights in a separate lane of same gel. References 1. Lacks, S.A., et al. (1979). Anal. Biochem. 100, Ro, G.M. and Maurer, W.D. (1986). In. IOP Publishing Limited, Bristol, England. pp Bollag, D.M., et al. (2002). Protein Methods. Second Edition. New York, N.Y. Wiley- Liss, Inc. pp.149. (Product # 20001). Ammonium Persulfate Catalyst for acrylamide gel polymerization. 6 (NH 4 ) 2 S 2 O 8 Ammonium Persulfate M.W Ammonium Persulfate 4 x 25 g $ 33 R: 36/37/38; S: 24/25, 27 TEMED Greater than 99% pure! C 6 H 16 N 2 TEMED M.W Specifications: Purity: >99.9% Refractive Index: Boiling Range: C Acrylamide polymerization reagent TEMED 25 ml $31 (N,N,N,N -Tetramethylethylenediamine) R: 11, 35, 37, 41; S: 26, 36/37/39, 45 For more product information, or to download a product instruction booklet, visit

6 Electrophoresis & Staining Flowchart 1. sample Buffers Choose MW markers Run Stain Post staining Urea A low UV-absorbing protein denaturant. Melting point: C Specification: A 280 < M Guanidine HCl Solution and Guanidine HCl 4,5 Ready-to-use, highly purified denaturants. H 2 N C NH 2 HCl NH Guanidine Hydrochloride M.W Free of UV-absorbing materials in range of nm Sharp UV cut-off spectrum with OD 260 less than 0.03 Typical metals: Cu 1 ppm; Fe 0.1 ppm; Pb 0.1 ppm; Zn 0.1 ppm Particulate-free, crystal-clear, colorless solution Excellent stability Excellent for washing affinity ligand columns (nonprotein ligands) 8 M Guanidine Hydrochloride Dilution Table Beginning with 10 ml of Pierce 8 M Guanidine HCl Solution (Product # 24115), dilution to indicated final volume will give stated molarity Urea 1 kg $26 R: 36/38; S: 26, 28 Molarity Volume of Final Volume 8 M Guanidine Molarity Volume 8 M 10 ml 3 M 26.7 ml 7 M 11.4 ml 2 M 40 ml 6 M 13.3 ml 1.5 M 52 ml 5 M 16 ml 1 M 80 ml 4 M 20 ml 0.5 M 160 ml References 4. Tanaka, S., et al. (1985). J. Biochem. 97(5), Wong, K.P., et al. (1971). Anal. Biochem. 40(2), Molar Guanidine HCl Solution 200 ml $147 Sequanal Grade R: 25, 36/37/38; S: 20/21, 26, Guanidine HCl 500 g $104 Crystalline, Sequanal Grade R: 25, 36/37/38; S: 20/21, 26, 28 Step 1 7 SDS (Sodium Dodecyl Sulfate) 6-8 When high resolution is key, this is ideal detergent. CH 3 (CH 2 ) 11 OSO 3 Na SDS M.W SDS (C 12 ) Greater than 99% alkyl sulfate Greater than 98% C 12 alkyl sulfate Contains a low level of hexadecyl sulfate C 16, which inhibits protein renaturation SDS (Lauryl) Unique distribution of carbon chain lengths is advantageous when resolving viral proteins during gel electrophoresis Can be used for renaturation after SDS-PAGE (if gels are treated according to procedure of Blank, et al. 8 to remove C 14 and C 16 alkyl sulfates) References 6. Maka, H.D., et al. (1977). Anal. Biochem. 81(1), Swaney, J.B., et al. (1974). Anal. Biochem. 58(2), Blank, A., et al. (1980). Federation Proceedings 39(6), Abstracts ABSC/TBS, Abstract No. 1285, SDS, C 12 Grade 500 g $119 (Sodium Dodecyl Sulfate, >98% C 12 ) R: 36/38; S: 26, SDS 100 g $41 (Sodium Dodecyl Sulfate, Lauryl) Typical Analysis: C 12 : 63.5%. C 14 : 29.5%, C 16 : 7.0% R: 22, 36/37/38; S: 23, 24/25, 36/37/ SDS 1 kg $143 (Sodium Dodecyl Sulfate, Lauryl) Typical Analysis: C 12 : 63.5%. C 14 : 29.5%, C 16 : 7.0% R: 22, 36/37/38; S: 23, 24/25, 36/37/39 Tel: or

7 Step 1 8 Precise Protein Gels Long shelf life short run time. Precise Protein Gels are cast in a durable plastic cassette using a neutral ph buffer that prevents polyacrylamide breakdown and results in a long shelf life. High-resolution staining and transfer of proteins is accomplished quickly on se 1 mm thick gels. Gels are individually packaged in an easy-to-open plastic pouch and are ready to run with no comb or tape to remove. The gels are available in both gradient and fixed concentrations and in 10-, 12- and 15-well formats. 12-month guarantee ensures consistent performance 45-minute run time provides results quickly Sample wells hold up to twice volume of Novex Brand gels (10-well=50 µl, 12-well=30 µl, 15-well=25 µl) Unique running buffer produces excellent separation and high-resolution protein bands Compatible with Laemmli sample buffer Compatible with standard mini-gel tanks so re is no need to purchase new equipment Stains quickly and with high sensitivity using coomassie and silver stains Transfers quickly and efficiently to nitrocellulose and PVDF membranes for Western blotting Migration Distance Migration Table Gel Percentage 8% 10% 12% 4%-20% 8%-16% Gel Specifications Cassette size 10 cm x 8.5 cm x 4.5 mm Gel size 8 cm x 5.8 cm x 1 mm Shelf life 12 4 C Running buffer Tris-HEPES-SDS Sample buffer Tris-HCl-SDS Compatible Gel Tanks BioRad Mini-PROTEAN II and 3 Novex XCell I, II and SureLock IBI Universal Protein System Hoefer Mighty Small II (SE 260/ SE 250) Hoefer Tall Mighty Small II (SE 280) Owl Road Runner and Penguin Owl Single-Sided Vertical System Percent # of Sample Sample Pkg. Product # Acrylamide Wells Well Volume Size Price % µl 10 gels $ % µl 10 gels $ % µl 10 gels $ % µl 10 gels $ % µl 10 gels $ % µl 10 gels $ % µl 10 gels $ % µl 10 gels $ % µl 10 gels $ % µl 10 gels $ % µl 10 gels $ % µl 10 gels $ % µl 10 gels $ % µl 10 gels $ % µl 10 gels $102 Running Buffer Precise Protein Gels use a unique Tris-HEPES-SDS running buffer to improve band resolution and reduce run-time. The buffer can be made according to recipe provided in instruction booklet or purchased premixed. Tris-HEPES-SDS Buffer A nonreducing buffer for use with Precise Gels BupH Tris-HEPES-SDS Running Buffer 10 pack $42 Each pack yields 500 ml of 100 mm Tris, 100 mm HEPES, 3 mm SDS, ph 8 ± 0.25 when dissolved in 500 ml distilled water (5 liters total). For more product information, or to download a product instruction booklet, visit visit

8 Electrophoresis & Staining Flowchart 1. sample Buffers Choose MW markers Run Step 2 sample Before a sample can be loaded onto a gel for analysis, it must be properly prepared. Depending on type, this may involve denaturing proteins, reducing any disulfide bonds, adjusting ionic strength and removing interfering contaminants. Samples may contain substances that interfere with obtaining a well-resolved protein band in. Substances such as guanidine hydrochloride and ionic detergents can result in protein bands that appear smeared or wavy in or on a Western blot. The PAGEprep Protein Clean-up and Enrichment Kit (Product # 26800) from Pierce removes se interfering components using an affinity resin that selectively binds n releases proteins. Using 20 µl of PAGEprep Resin, a protein sample (5-400 µl) can be purged of any contaminants in only 10 minutes. This is much faster than dialysis or ultrafiltration and yields higher protein recoveries while concentrating sample. Stain Post staining Step 2 Step 2 sample 9 PAGEprep Protein Clean-Up and Enrichment Kit Better bands in minutes! Eliminates lane artifacts caused by incompatible contaminants (e.g., salts and chaotropic agents) to provide reproducible results on SDS-PAGE analysis Faster than ultrafiltration devices enriches dilute protein solutions by 20-fold in less than 10 minutes for SDS-PAGE analysis Remove dyes, reducing agents, detergents, sugars, glycerol, guanidine, urea and ammonium sulfate without dialysis Simple, easy protocol using microcentrifugation Unique PAGEprep Method can process samples as small as 0.5 µl Separate proteins from chaotropic agents, detergents, DNA/RNA, lipids and salts with new PAGEprep Protein Clean-Up and Enrichment Kit. Guanidine hydrochloride, low ph and ammonium sulfate are known to be incompatible with typical electrophoretic running buffers. Mixing protein samples containing 6 M guanidine HCl with Laemmli Buffer, for example, results in a sample that turns into a viscous mess. The sample is n unable to run efficiently on a denaturing gel (SDS-PAGE). Fortunately, this same sample can be cleaned-up easily using PAGEprep Kit by removing guanidine HCl and producing an electrophoresis-ready sample. Sample concentration is anor critical factor for proper SDS-PAGE analysis using standard staining methods. Protein samples that are too dilute to be detected reliably using traditional methods can now be concentrated rapidly using PAGEprep Kit and directly analyzed with SDS-PAGE. The PAGEprep Kit uses a unique resin of modified The Protein Protocols Handbook A must for anyone working with proteins. This handbook details 145 highly successful analytical methods for protein and peptide analysis. Featured protocols contain detailed instructions, troubleshooting tips, alternative procedures, informative explanations and timesaving techniques. Walker, J.M., Ed., Published by Humana Press, 1996, 809 pages, Comb-bound Product #: Price: $125 Tel: or

9 Step 2 sample 10 diatomaceous earth that quickly binds protein under an organic phase of DMSO. Simply combine between µl of your sample with DMSO and mix with 20 µl of PAGEprep Resin slurry in a microcentrifuge tube. Just 20 µl of resin can bind over 20 µg of protein. All proteins will bind to resin while contaminating chemicals are washed away during centrifugation. Elute sample in 50 µl of Elution/Gel Loading Buffer and sample is ready for electrophoresis. The unique pink tracking dye in buffer indicates when electrophoretic run is complete. The entire PAGEprep Protocol can be completed in less than 10 minutes. samples for SDS-PAGE analysis from: Inclusion bodies solubilized in guanidine HCl Column fractions containing low-ph buffers, thiocyanate or urea Proteins precipitated in ammonium sulfate Solutions of dilute protein Table 2. Sample Preparation Methods Comparison PAGEprep Kit Dialysis Ultrafiltration Sample Size 200 µl 200 µl 200 µl Protein Recovery 70-85% 70-90% 70-90% Processing Time <10 minutes 20 minutes hours minutes Concentrating Yes No Yes Comparison of samples containing 6 M guanidine HCl mixed with Laemmli Loading Buffer. A precipitate forms when 6 M guanidine HCl is mixed with Laemmli Loading Buffer. After PAGEprep Treatment to remove guanidine HCl, no precipitate is formed when protein sample is mixed with Laemmli Loading Buffer. Untreated PAGEprep Kit-Treated m s m s m s m s Removal of detergent from cell lysis fractions by PAGEprep Kit. Rat C6 cells were lysed and a membrane protein fraction isolated using Mem-PER Eukaryotic Membrane Protein Extraction Reagent (Product # 89826). Membrane (m) and hydrophilic (s) cell fractions were separated by SDS-PAGE (4-20% gradient gel) with or without prior treatment using PAGEprep Protein Clean-Up and Enrichment Kit. Western blot analysis was performed using an antibody against cytochrome oxidase subunit 4 (Cox4) and SuperSignal West Femto Chemiluminescent Substrate (Product # 34095). PAGEprep Kit-treated samples showed better band resolution than samples that were untreated and still contained detergent Dilute solution of BSA concentrated before SDS-PAGE analysis using PAGEprep Protein Clean-Up and Enrichment Kit. Lane 1 contains TriChromRanger Prestained Protein Molecular Weight Marker (Product # 26691). Lane 2 shows 10 µl of 0.1 mg/ml BSA (1 µg). Lane 3 shows eluted fraction after 10 µl of 0.1 mg/ml BSA was processed with PAGEprep Protocol. Lane 4 shows eluted fraction after 200 µl of 0.1 mg/ml BSA was processed with PAGEprep Protocol PAGEprep Protein Clean-Up Kit $104 and Enrichment Kit Sufficient reagents for preparing 50 samples. Includes: Modified Diatomaceous Earth (mde) 50% slurry Elution/Gel Loading Buffer Purified DMSO R: 36/37/38; S: 36/37/39 1 ml 2.5 ml 25 ml For more product information, or to download a product instruction booklet, visit visit

10 Electrophoresis & Staining Flowchart 1. 2-D Gels sample Buffers Choose MW markers Isolating and extracting proteins may result in charged buffer components that interfere with IEF in first dimension of 2-D electrophoresis. To address this, Pierce offers four unique kits for 2-D sample preparation: 2-D Sample Prep for Membrane Proteins (Product # 89864) 2-D Sample Prep for Nuclear Proteins (Product # 89863) 2-D Sample Prep for Soluble Proteins (Product # 89865) 2-D Sample Prep for Insoluble Proteins (Product # 89866) These 2-D Sample Preparation Kits contain mini-desalting spincolumns for exchanging small sample sizes (<400 µl) directly into a 2-D sample buffer supplied in kits. The protein sample is effectively concentrated as it is desalted. This sample can be directly applied to IEF gel. This assures that 2-D gel results are consistent and proteins migrate properly in second dimension. 9,10 The figure to right shows a comparison of two samples, one prepared with 2-D Sample Preparation Kit for Insoluble Proteins and or left untreated. The desalting treatment alleviates interference from salt fronts and clearly allows more data to be seen for comparing protein expression differences among samples. MW (K) Run A. Treated B. Untreated Stain 4 pl 7 References 9. Rabilloud, T., et al. (1997). Electrophoresis 18, Lanne, B., et al. (2001). Proteomics 1, Post staining More Protein, Data Retained Less Protein, Missing Data Step 2 sample 2-D Sample Prep Kit for Membrane Proteins Suited for membrane protein fractionation along with sample cleanup. Streamline membrane protein extraction with 2-D sample preparation. Membrane proteins are isolated, concentrated and exchanged into 2-D sample buffer without precipitation. Ideal for isolating low-abundance membrane proteins. Requires no precipitation eliminates difficult resolubilization steps Isolates efficiently isolates µg of membrane proteins Concentrates and cleans up membrane proteins are concentrated up to six-fold and excess detergent is removed using 2-D PAGEprep Resin Buffer exchanges membrane proteins into 2-D Sample Buffer maintains proteins in solution throughout desalting process and allows cleaned-up sample eluate obtained to be electrophoresed in its entirety Works quickly membrane proteins are prepared in less than one hour and are cleaned up in minutes Contains thiourea in 2-D Sample Buffer increases protein solubility and improves protein resolution on 2-D gels D Sample Prep for Membrane Kit $329 Proteins Kit Sufficient reagents for 25 applications. Includes: Mem-PER Eukaryotic Membrane Protein Extraction Kit: Mem-PER Cell Lysis Reagent 5 ml Mem-PER Buffer 12.5 ml Mem-PER Membrane Protein 20 ml Solubilization Reagent 2-D Sample Buffer for Membrane Proteins: 2-D Sample Buffer for Membrane 18 ml Proteins Component A 2-D Sample Buffer for Membrane 16.5 g Proteins Component B 2-D PAGEprep Protein Clean-up and Enrichment Kit: 2-D PAGEprep Protein Binding Resin 0.5 ml Binding/Wash Buffer Stock Solution 50 ml DMSO Protein Desalting Spin Columns 25 columns Elution Buffer 1.25 ml Spin cups 25 cups Collection tubes 50 tubes R: 25, 36, 37/38, 40, 41, 45, 51/53; S: 1/2, 24, 26, 27, 36/37, 39, 45, 46, 53, Tel: or

11 Step 2 sample 12 2-D Sample Prep Kit for Nuclear Proteins Suited for nuclear protein fractionation along with sample cleanup. Streamlines nuclear protein extraction with 2-D sample preparation. Nuclear proteins are isolated, concentrated and exchanged into 2-D sample buffer without precipitation. Removes small charged contaminants that interfere with 2-D electrophoresis reduces time for isoelectric focusing and prevents loss of data on 2-D gels due to salt fronts Buffer exchanges nuclear proteins into 2-D Sample Buffer concentrates protein by increasing amount of protein that can be applied to an IPG strip and maintains proteins in solution throughout desalting process Uses NE-PER Nuclear and Cytoplasmic Reagents prepares a highly purified nuclear protein extract Streamlines nuclear protein extraction with 2-D sample preparation contains a faster and more efficient protocol than two procedures performed separately Contains thiourea in sample buffer increases protein solubility and improves protein resolution on 2-D gels Desalts faster than existing 2-D sample prep kits allows multiple samples to be processed in less than 15 minutes instead of one plus hours required for precipitation and dialysis D Sample Prep for Nuclear Kit $299 Proteins Kit Sufficient reagents for 25 applications. Includes: NE-PER Nuclear and Cytoplasmic Extraction Reagents: Cytoplasmic Extraction 5 ml Reagent I (CER I) Cytoplasmic Extraction ml Reagent II (CER II) Nuclear Extraction 2.5 ml Reagent (NER) 2-D Sample Buffer for Nuclear Proteins: 2-D Sample Buffer for Nuclear 18 ml Proteins, Component A 2-D Sample Buffer for Nuclear 16.5 g Proteins, Component B Protein Desalting Spin Columns 25 columns R: 25, 40, 51/53; S: 1/2, 36/37 2-D Sample Prep Kit for Soluble/Insoluble Proteins Suited for soluble and insoluble cellular protein fractionation along with sample cleanup. The Sample Prep Kit for Soluble Proteins is designed for whole cell and tissue extracts as well as for fractionated proteins in which proteins of interest are predominantly hydrophilic. The Sample Prep Kit for Insoluble Proteins is designed for proteins that are more difficult to solubilize including cell extracts containing larger and/or more hydrophobic proteins and proteins that tend to aggregate. Removes small charged contaminants that interfere with 2-D electrophoresis reduces time required for isoelectric focusing and prevents loss of data on 2-D gels due to salt fronts Buffer exchanges proteins into 2-D Sample Buffer concentrates protein by increasing amount of protein that can be applied to an IPG strip AND maintains proteins in solution throughout desalting process Works faster than existing 2-D sample prep kits processes multiple samples in less than 15 minutes instead of one plus hours required for precipitation and dialysis Contains thiourea in 2-D Sample Buffer for Insoluble Proteins increases protein solubility and improves protein resolution on 2-D gels D Sample Prep for Soluble Kit $199 Proteins Kit Sufficient reagents for 25 applications. Includes: 2-D Sample Buffer for Soluble Proteins: 2-D Sample Buffer for Soluble 19.5 ml Proteins, Component A 2-D Sample Buffer for Soluble 15 g Proteins, Component B Protein Desalting Columns 25 columns D Sample Prep for Insoluble Kit $199 Proteins Kit Sufficient reagents for 25 applications. Includes: 2-D Sample Buffer for Insoluble Proteins: 2-D Sample Buffer for Insoluble 18 ml Proteins, Component A 2-D Sample Buffer for Insoluble 16.5 g Proteins, Component B Protein Desalting Columns 25 columns R: 25, 40, 51/53; S: 1/2 For more product information, or to download a product instruction booklet, visit