ProteoEnrich CAT-X SEC Kit Merck: Lichrospher Composition. Silica Particle size. 25 µm particles with 6 nm pore size Ligand SO 3

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1 Novagen User Protocol TB428 Rev. A of 6 ProteoEnrich TM CAT-X SEC Kit About the Kit ProteoEnrich CAT-X SEC Kit Description The ProteoEnrich CAT-X SEC Kit provides a highly specific method to enrich the proteome of a biological sample such as bacterial extracts, body fluids and crude tissue samples for low molecular weight proteins. The method is based on a unique resin that allows proteins and polypeptides with a globular size of less than 20 kda to penetrate the resin pores and bind to the surface of the inner pores according to their net charge while larger proteins flow through the resin-filled cartridge. After a salt gradient or single step elution and excess salt removal, the sample is ready for downstream analysis by mass spectrometry, SELDI, or array-based analysis. The kit contains two CAT-X SEC cartridges with Luer Lock adapters, optimized buffers, and BSA. The cartridges can be used as syringe-tip filters or as liquid chromatography devices. Each cartridge binds up to 5 mg complex protein mixture and can be reused at least 10 times. Components Solid support Merck: Lichrospher Composition Silica Particle size 25 µm particles with 6 nm pore size Ligand SO 3 H and Silanol groups Static capacity 5mg protein (plasma) per ml/resin Capacity for fractionation Dependent on sample type ph stability range 2 11 Storage 4 C 2 CAT-X SEC Cartridges (with Luer Lock Adaptor) 90 ml 10X CAT-X SEC Bind/Wash Buffer (250 mm potassium phosphate buffer, ph 3.0) 90 ml CAT-X SEC Elute Buffer [2 M NaCl, 25 mm potassium phosphate buffer, acetonitrile (10% v/v) ph 3.0] 3 ml BSA, 100 mg/ml (nuclease- and protease-free, 0.09% Sodium Azide) Storage Store all components at 4 C. Protect the CAT-X SEC Cartridges from freezing. Freezing and thawing will damage the resin. Additional reagents and equipment Syringes for manual use. Syringes with Luer Lock Adaptors are recommended. Deionized water (resistance > 18 mω) EMD Biosciences, Inc., an affiliate of Merck KGaA, Darmstadt,. All rights reserved. The Novagen name is a registered trademark of EMD Biosciences, Inc. in the United States and in certain other jurisdictions. ATP-Binders, D-Tube, Non-Interfering Protein Assay, and ProteoEnrich, are trademarks of EMD Biosciences, Inc. Fractogel, and ProteoExtract are registered trademarks of Merck KGaA, Darmstadt,. NuPAGE is a registered trademark of Invitrogen Corp. A Brand of EMD Biosciences, Inc., an Affiliate of Merck KGaA, Darmstadt, FOR RESEARCH USE ONLY. NOT FOR HUMAN OR DIAGNOSTIC USE.

2 User Protocol TB428 Rev. A of 6 Figure 1. The principle of SEC-based multidimensional liquid chromatography is illustrated. The CAT-X SEC Cartridge material features an external hydrophilic surface, that allows efficient exclusion of high molecular weight proteins, and an internal cation exchange surface that retains low molecular weight proteins through electrostatic interactions. Figure 1 Figure 2. The enrichment of low molecular weight components from liver tissues using SELDI IMAC analysis is demonstrated: Protein extraction was performed using the ProteoExtract Partial Proteome Extraction Kit (P-PEK; ) according to the user protocol. 1.5 mg extracted sample was loaded on a conditioned CAT-X SEC Cartridge. LC was performed using binding buffer: 25 mm potassium phosphate ph 3.0. Elution buffer: 2 M NaCl 25 mm potassium phosphate, ph 3.0, 10 % (v/v) acetonitrile. Flow rate 0.1 ml/min. After LC prefractionation 150 ml was loaded on a SELDI IMAC chip. After chip loading, SELDI was performed according to user protocol. Figure 3. An example of the enrichment of small size protein fraction from TGF-β stimulated human fetal lung fibroblast cells is provided. One-Dimensional-PAGE (4 12% NuPAGE ) fractions from solid-phase extraction, flow through (lane 1), wash fraction (lane 2) and eluted fraction (lane 3). 100 µg TGF- β stimulated HFL-1 cells was loaded to the column. [1] Figure 3 Figure 2

3 User Protocol TB428 Rev. A of 6 ProteoEnrich CAT-X SEC Kit The CAT-X SEC Cartridges can be used either manually or with a liquid chromatography (LC) instrument (See Figure 4, below). When used manually, the CAT-X SEC Cartridges are operated with a syringe. When used under LC-instrument control, connect the CAT-X SEC Cartridge to a 1/16-inch port of a standard HPLC/FPLC instrument. All connections must be finger tight. Application of too much force when closing the connections will impair the CAT-X SEC Cartridge function and may cause leakage. The volume of the CAT-X SEC Cartridge is one milliliter and can be used to fractionate at least one milligram crude protein extract. One cartridge can be used to bind greater than one milligram crude protein, however, optimal resolution is obtained when the sample contains approximately five milligrams. For larger samples, multiple CAT-X SEC Cartridges can be linked using the supplied Luer Lock Adaptor. Manual use 1. Remove black plug from upper end of CAT-X SEC Cartridge by turning counterclockwise. 2. Connect provided Luer Lock Adaptor by screwing it into the stopper. 3. Remove transparent lower plug. The cartridge is ready for manual operation with a syringe. A second cartridge may be connected to increase the binding capacity if desired (See Figure 4, below). Use with LC-instrument 1. Remove transparent lower plug from the CAT-X SEC Cartridge. 2. Connect provided Luer Lock Adaptor by screwing it into the fitting at end of CAT-X SEC Cartridge. 3. Insert CAT-X SEC cartridge/adaptor setup into a 1/16-inch port of LC-instrument valve. Up to three cartridges may be connected to increase binding capacity (See Figure 4, below). Figure 4. Syringe tip filter and LC-instrument CAT-X SEC Cartridge preparation.

4 User Protocol TB428 Rev. A of 6 General Considerations Effective separation of small positively charged proteins can be achieved with alternative buffers and ph. Buffer ph should be chosen so that proteins of interest have a net positive charge. The operating ph should be at least 1 unit below the pi of the protein(s) of interest. Suitable buffers include sodium acetate-acetic acid (pka ), citric acid-sodium citrate (pka ), and glycine-hcl (pka ). Generally, increasing the buffer ph will reduce the total number of proteins adsorbed to the resin from a crude extract. Bind/wash buffer should have an ionic strength of 25mM. Elute buffer should contain the same buffering ion and molarity as the bind/wash buffer plus 2 M NaCl and 10% acetonitrile. Acetonitrile may be omitted from the elute buffer if the organic solvent negatively impacts the quality or activity of the desired protein eluate. Sample preparation ProteoEnrich CAT-X SEC cartridges can be used to enrich small analytes from crude protein extracts, such as bacterial or tissue extracts, and body fluid samples (serum or plasma). Body fluid samples require no special pretreatment. In general, the ProteoEnrich CAT-X SEC Kit is compatible with lysis buffers (e.g., urea or sodium chloride < 50 mm) that do not contain ionic detergents or high salt concentrations. The procedure is compatible with ProteoExtract Subcellular Proteome Extraction Kit (S-PEK; ), with the exception of S-PEK fraction 4, and ProteoExtract Native Membrane Protein Extraction Kit (M-PEK; ) fractions. Samples from ProteoExtract Complete Proteome Extraction Kits (C-PEK; Cat. Nos , , ), ProteoExtract Partial Proteome Extraction Kit (P-PEK; ), and S-PEK fraction 4 cannot be used without first reducing the salt concentration. If the salt concentration is greater than 50 mm in the undiluted sample, the sample should be dialyzed into an appropriate buffer (e.g., 25 mm potassium phosphate buffer, ph 3.0) before proceeding with the fractionation protocol. The D-Tube Maxi Dialyzers (Cat. Nos , 71509, 71510) are an extremely convenient system for dialysis of biological sample volumes up to 3 ml. Following protein extraction, samples should be cleared by centrifugation (e.g., 5 min at 14,000 g) or filtration through a 0.45 µm low protein binding filter to avoid clogging the cartridge. Fractionation protocol This section describes fractionation by manual operation and by LC-instrument controlled runs. Protein fractionation takes approximately 20 minutes using the manual protocol. The time required for LC instrument-driven fractionation will vary depending upon the program used. The CAT-X SEC Cartridge is stable up to a pressure drop of five bars. Fractionation can be performed at 4 C or at room temperature. The volume given in the following protocols is for one cartridge. If using multiple cartridges adjust volumes accordingly. Fractionation by manual operation 1. Dilute protein sample with equal vol 10X CAT-X SEC Bind/Wash Buffer and 8 vol deionized water. The following is an example for a 50 µl sample: 50 µl Protein sample (approximately 5 mg protein) 50 µl 10X CAT-X SEC Buffer 400 µl Deionized water 500 µl total volume 2. Prepare 15 ml 1X CAT-X SEC bind/wash buffer by diluting 10X CAT-X SEC Bind/Wash Buffer with deionized water. 3. Optional: If the total amount of target protein in the sample is low, prepare 3 ml 10 mg/ml BSA in 1X bind/wash buffer. 4. Fill syringe with 10 ml 1X CAT-X SEC bind/wash buffer prepared in Step 2. Remove any trapped air from the syringe, and connect to the cartridge. 5. Equilibrate cartridge with 2 ml 1X CAT-X SEC bind/wash buffer by applying gentle pressure to plunger of syringe. Discard flow-through. Remove syringe.

5 User Protocol TB428 Rev. A of 6 6. Optional: If the total amount of target protein in the sample is low, condition cartridge with 1 ml 10 mg/ml BSA solution in 1X CAT-X SEC bind/wash buffer followed by 5 ml 1X CAT-X SEC bind/wash buffer. Repeat one time. 7. Fill a clean syringe with diluted sample prepared in Step 1. Remove any trapped air from the syringe and connect to the cartridge. 8. Gently depress the plunger to push the diluted sample through the cartridge at a flow rate of approximately 30 drops per minute. This will allow for sufficient contact time between the sample and the resin. Collect the flow-through as Unbound fraction and store at 4 C for further analysis. 9. Remove syringe that had contained sample and connect syringe containing 1X CAT-X SEC bind/wash buffer (filled in Step 4). Wash cartridge with 3 ml 1X CAT-X SEC bind/wash buffer by applying gentle pressure to the plunger at a flow rate of approximately 30 drops per minute. Collect flow-through as Wash fraction, and store at 4 C for further analysis. 10. Disconnect syringe and remove any remaining 1X CAT-X SEC bind/wash buffer from syringe. Fill new syringe with 3 ml CAT-X SEC Elute Buffer. Connect syringe to cartridge and push buffer through cartridge by applying gentle pressure to the plunger. Collect eluate and store at 4 C for further analysis. Note: For preparation of fractions for downstream analysis, see Additional Guidelines, page The cartridges can be re-equilibrated and reused. See Cartridge regeneration and cleaning, page 6. Fractionation by LC-instrument controlled operation 1. Dilute the protein sample with an equal volume of 10X CAT-X SEC Buffer and 8 vol deionized water. The following is an example for a 50 µl sample: 50 µl Protein sample (~5 mg protein) 50 µl 10X CAT-X SEC Buffer 400 µl Deionized water 500 µl total volume 2. Prepare a sufficient volume of 1X CAT-X SEC bind/wash buffer. 3. Optional: If the total amount of target protein in the sample is low, prepare 3 ml 10 mg/ml BSA in 1X bind/wash buffer. 4. Connect the CAT-X SEC Cartridge or cartridge assembly as indicated on page Equilibrate your LC system with these buffers per manufacturer instructions. 6. CAT-X SEC cartridges should be operated with flow rates between 0.1 ml/min and 1 ml/min for efficient protein enrichment. A flow rate of 0.5 ml is generally recommended when fractionating up to 1 mg protein sample per CAT-X SEC Cartridge. If loading higher amounts of protein, we recommend lowering the flow rate to 0.1 ml/min. 7. Use the following guidelines for programming your LC instrument for liquid chromatography: a) Equilibrate CAT-X SEC cartridge(s) with 3 vol 1X CAT-X SEC bind/wash buffer. b) Optional: If the total amount of target protein in the sample is low, condition CAT-X SEC cartridge(s) with 10 mg/ml BSA in 3 vol 1X CAT-X SEC bind/wash buffer. Wash with 1X CAT-X SEC bind/wash buffer until UV is nearly baseline (0). Repeat one time. c) Optional: After conditioning (Step 7. b), wash with 3 ml CAT-X SEC Elute Buffer. Follow with 3 ml wash with 1X CAT-X SEC bind/wash buffer. d) Inject diluted sample. Collect flow-through as Unbound fraction and store at 4 C for further analysis. e) Wash the cartridge(s) with 3 vol 1X CAT-X SEC bind/wash buffer. Collect the flowthrough as Wash fraction and store at 4 C for further analysis. f) Elute fractions step-wise using 3 vol CAT-X SEC Elute Buffer. Collect volume controlled eluate fractions. g) The cartridges can be re-equilibrated and reused. See Cartridge regeneration and cleaning, page 6.

6 User Protocol TB428 Rev. A of 6 Cartridge regeneration and cleaning With proper regeneration and cleaning, the ProteoEnrich CAT-X SEC Cartridges can be used at least ten times. Regeneration should be performed immediately following fractionation or purification. Wash cartridges with 3 ml 2 M NaCl in CAT-X SEC Elute Buffer, followed by 5 ml 1X CAT-X SEC bind/wash buffer. Store at 4 C in 20% ethanol. Additional Guidelines Protein Quantification Absorbance at 280 nm can be used to estimate the protein concentration. For more accurate protein quantification, any method not influenced by the presence of acetate buffer can be used, including the BCA Protein Assay Kit ( 71285) and Non-Interfering Protein Assay Kit ( ). Enrichment The eluted sample can be used to enrich phosphoproteins using ProteoEnrich ATP-Binders Kit ( 71438), or phosphopeptides using ProteoExtact Phosphopeptide Capture Kit ( ). One-dimensional gel electrophoresis Mix the sample with an equal volume of 2X SDS Sample Buffer (4X SDS Sample Buffer, 70607). Heat samples to C for 5 min and cool to room temperature prior to loading. One-dimensional gel electrophoresis can usually be performed without concentration of the fractionated sample. Two-dimensional gel electrophoresis Two-dimensional gel electrophoresis of fractionated samples yields optimal results after removing excess salts. The D-Tube Maxi Dialyzers (Cat. Nos , 71509, 71510) are an extremely convenient system for dialysis of biological sample volumes up to a 3 ml. The ProteoExtract Protein Precipitation Kit ( ) is also available for fast and efficient concentration and clean up of protein samples. Reference 1. Bratt, C. and et.al., J Chromatogr A, : p