GST 蛋白纯化 High-Affinity GST Resin genescript TO P

High-Affinity GST Resin Technical Manual No. TM0185 Version 10252005 I Description….…………………………………………………………………….…………. 1 II Specifications….……………………………………………………………………….……. 1 III Key Features ………………………………………………………………………………. 1 IV Related Products …………………………………………………………………………. 1 V Storage ……………………………………………………………………………………. 2 VI GST-Fused Protein Purification Protocol …………………….……………………..…… 2 VII Troubleshooting …………………………………………………………………………... 3 VIII Order Information……………………………………………………………………………. 4 I. DESCRIPTION GenScript High-Affinity GST Resin (L00206) is designed for the rapid, single-step purification of glutathione S- transferases, glutathione-dependent proteins and recombinant derivatives of glutathione S-transferase, including glutathione S-transferase (GST) fusion proteins expressed in E. coli, insect cells and mammalian cells. GST fusion proteins can be purified directly from bacterial lysate using High-Affinity GST Resin. High-Affinity GST Resin (50% slurry) has a protein binding capacity of > 6 mg GST/ml packed resin. II. SPECIFICATIONS Resin volume 20 ml of 50% slurry Ligand Glutathione Capacity > 6 mg horse liver GST/ml packed resin Matrix Cross-linked agarose Average particle size 90 µm III. KEY FEATURES ♣ Easy to perform: simple and rapid procedure to purify GST-fusion protein. ♣ High capacity: > 6 mg horse liver GST/ml medium. ♣ Stability: no obvious decrease of the binding capacity after reusing three times. IV. RELATED PRODUCTS GenScript also provides two kits to facilitate the expression and purification of GST fusion proteins: L00207 GST Fusion Protein Purification Kit L00208 Protein Expression and Purification Kit GenScript Corporation Tel: 732-885-9188 Fax: 732-210-0262 www.genscript.com email: info@genscript.com Components L00206 L00207 L00208 GST Resin 20 ml 20 ml 20 ml Columns 5 empty columns 5 empty columns Glutathione, reduced 5 X 0.154 g 5 X 0.154 g Enterokinase 2 X 100 IU Expression vector pGS-21a Manual TM0185 TM0185 TM0186 High-Affinity GST Resin 2 GenScript Corporation Tel: 732-357-3839 Fax: 732-210-0262 www.genscript.com email: info@genscript.com V. STORAGE Store High Affinity GST Resin in 20% ethanol at 4°C, do not freeze. VI. GST-FUSED PROTEIN PURIFICATION PROTOCOL A. Preparation of Cell Extract 1. Harvest cells by centrifugation at 3,000 g at 4oC for 10 min, remove and discard the supernatant. Resuspend the cell pellet in 3 mL ice-cold PBS Buffer per 50 mL culture and centrifuge at 3,000 g at 4oC for 10 min. Remove and discard the supernatant. 2. Freeze the cell pellet at -80 ºC for 1 hour (This is also a convenient point to stop and one can continue the procedure later). 3. Thaw cell pellet on ice and re-suspend cells in 3 mL of ice-cold PBS Buffer per 50 mL culture. If desired, add appropriate additives, such as non-ionic detergents (NP-40) or protease inhibitors (PMSF). 4. Break the cells by brief pulses of sonication on ice until the sample is no longer viscous. 5. Centrifuge at 12,000 g at 4 ºC for 10 min and carefully transfer the supernatant (Soluble Fraction) to a clean and pre-chilled tube and resuspend pellet (Insoluble Fraction) with 3 mL of ice-cold PBS Buffer per 50 mL of E. coli culture. 6. Aliquot 10 µL samples from both Soluble and Insoluble Fractions for SDS-PAGE Analysis (by adding equal volume of 2 x SDS sample loading buffer, boil for 5 min and run SDS-PAGE to determine the amount and solubility of the GST-fusion protein). Note: a. The binding of GST or GST-fusion protein to High Affinity GST Resin is not affected by 1% Triton X-100, 1% Tween-20, 1% CTAB, 10 mM DTT, 0.03% SDS, or 0.1% NP-40. These chemicals may be used to reduce non-specific binding. b. If the target GST-fusion protein forms inclusion body (insoluble protein), inclusion body has to be properly solubilized and refolded prior to purification). B. Purification of Recombinant GST-Fusion Protein 1. Shake gently the bottle containing the High-Affinity GST Resin until all the resin is completely in suspension. 2. Transfer an appropriate amount of resin (50% slurry) to a disposable column (included in Kit L00207 and L00208) using a pipet. Usually 1 ml of resin (from 2 ml of 50% slurry) can bind more than 6 mg of GST protein. 3. Wash the GST resin with 10 bed volumes of cold (4°C) PBS. 4. Apply clear solution (sonicate, etc) containing GST-fusion protein in PBS to the equilibrated column with the flow rate at 10-15 cm/h. 5. Add PBS to wash the column just after all the protein solution get into the column, use 20 bed volumes of PBS for wash. Protease inhibitors such as PMSF are better added to the wash solution to inhibit protease activity. 6. Elute the fusion protein with 10-15 bed volumes of freshly made 10 mM Glutathione Elution Buffer (0.154 g of reduced glutathione dissolved in 50 ml of 50 mM Tris-HCl, pH 8.0.). 7. Monitor elution of the fusion protein using absorbance readings at 280 nm. 8. Aliquot 10-20 µL of supernatant containing GST-fusion protein, flow through, wash and the eluted protein, respectively, and analyze all the samples by running SDS-PAGE to confirm the presence of the target protein. An example was shown in Figure 1. 9. Pool eluted fractions containing target protein. Remove free glutathione by dialysis at 4 ºC against a buffer of choice or by using a G15 Sephadex desalt column. C. Regeneration and Storage of High Affinity GST Resin GenScript High-Affinity GST Resin can be reused to purify the same protein three times without regeneration. If the target GST-fusion protein is different, however, High-Affinity GST Resin must be regenerated using the following protocol: 1. Wash the column with 2 bed volumes of 0.1 M Tris HCl + 0.5 M NaCl, pH 8.5. 2. Wash the column with 2 bed volumes of 0.1 M sodium acetate + 0.5 M NaCl, pH 4.5. 3. Re-equilibrate the column with 3-5 bed volumes of 1X PBS. 4. For long-term storage, the resin should be stored in 20% ethanol at 4 ºC. High-Affinity GST Resin 3 GenScript Corporation Tel: 732-357-3839 Fa 1 2 3 4 1 Total protein 2 Flow through 3 Wash 4 Elute VII. Troubleshooting The table below is guideline for troubleshooting. Problem Probable Cause Solution The yield of the purified fusion protein is low or undetectable The fusion protein forms inclusion body. The fusion protein does not bind to GST Resin efficiently. The fusion protein does not contain active GST. The fusion protein is degraded by protease. The fusion protein is not efficiently eluted from High Affinity GST Resin. Grow bacteria at low temperature (20-30 ºC), or reduce final concentration of IPTG to 0.1 mM for protein induction, or reduce the induction time. Properly dissolve and refold the inclusion body prior to the purification. Use batch method for purification. Incubate clear solution (sonicate, etc) containing GST-fusion protein with GST Resin for 2 hours or longer (such as overnight) and then load the mixture onto the column. Use mild sonication condition or other lysis method, such as lysozyme so that GST is not denatured. Add appropriate protease inhibitors such as PMSF in the lysis solution and wash solution. Increase elution time or Increase the concentration of fer. Fig.1. Purification profile using High-Affinity GST Resin - glutathione to 15 mM or higher in the Elution Buf x: 732-210-0262 www.genscript.com email: info@genscript.com Adjust the pH of the Elution Buffer to 8.0-9.0 without increasing the glutathione concentration. Add Triton X-100 (0.1%, final concentration) or Noctylglucoside (2%, final concentration) or NaCl (0.1 - 0.2 M, final concentration) to the Elution Buffer. High-Affinity GST Resin 4 GenScript Corporation Tel: 732-357-3839 Fax: 732-210-0262 www.genscript.com email: info@genscript.com Multiple bands observed in the eluted protein The fusion protein is degradated by protease. Some host proteins, such as chaperonins, may interact with the fusion protein. Over-sonication will cause some protein to bind to the fusion protein. Some protein will bind to the fusion protein or beads non- specifically. Add appropriate protease inhibitors (or inhibitor cocktails) such as PMSF in the lysis solution and wash solution. Add DTT (5 mM, final concentration) in the Wash Buffer. Incubate the recombinant protein solution in Chaperonin Buffer (2 mM ATP, 10 mM MgSO4, 50 mM Tris-HCl) at 37 ºC for 10 min prior to the purification. Use milder sonication condition or another lysis method. Optimizing the wash conditions. Detergents such as 1% Triton X-100, 1% Tween-20, 0.03% SDS, or 0.1% NP-40 may be used to reduce non-specific binding. Salt concentration in the wash solution can also be optimized to reduce non-specific binding. VIII. ORDER INFORMATION High Affinity GST Resin Catalog Number: L00206 For Research Use Only. GenScript Corporation 120 Centennial Ave., Piscataway, NJ 08854 Tel: 732-885-9188, 732-357-3839 Fax: 732-210-0262, 732-885-5878 Email: info@genscript.com Web: http://www.Genscript.com High-Affinity GST Resin Technical Manual No. TM0185 Version 10252005 � II. SPECIFICATIONS The table below is guideline for troubleshooting. Problem Probable Cause Solution Use mild sonication condition or other lysis method, such as lysozyme so that GST is not denatured. Multiple bands observed in the eluted protein Add appropriate protease inhibitors (or inhibitor cocktails) such as PMSF in the lysis solution and wash solution. GenScript Corporation