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1、精品文档GST 融合蛋白纯化方法 Purification of GST Fused ProteinsAbstract:Many people have ven ted out frustratio n over in soluble GST-fused prote ins. This is a protocolfor en zymatically active soluble GST-fused protei ns. All GST-fused prote ins are ren dered soluble with this tech nique though en zyme activi
2、tiy can range from 30-90%.Materials and Reage nts1. STE Buffer10 mM Tris-HCl, pH 8.01 mM EDTA150 mM NaCl2. Lysozyme soluti on10 mg/ml in water (make fresh)3. PBS4. Elution Buffer50 mM Tris.Cl, pH 9.020 mM GSH5. 10% Sarkosyl in STE Buffer6. 10% Triton X-100 in STE Buffer7. 1 M DTT8. 100 mM IPTGProced
3、ureDay 11. Set up an overnight culture in 50 ml 2XTY with 150 mg/ml of ampicillin.Day 21. Seed 5 ml of overnight culture to 500 ml 2XTY with 150 mg/ml of ampicilli n.2. Grow at 37 C to an A600 of 0.6 to 0.8.3. Induce with 0.1 mM to 2 mM of IPTG. Grow for 3 hr at 37C or grow overnight at room tempera
4、ture.Lower IPTG concen trati ons and lower grow ing temperatures tend to produce greater solubility at the expe nse of yield.4. Pellet cells by cen trifugi ng at 3000g, 4 oC for 10 min. Deca nt media and resuspe nd cells in 30 ml ice-coldPBS to wash. Tran sfer to a 40-ml Oak Ridge tube and cen trifu
5、ge at 3000 g, 4oC for 10 min. Deca nt PBS.5. This is a convenient point to stop and to store pellets at -80oC. Else continue to lyse cells.6. Thaw pellet on ice if cells are froze n else proceed to the n ext step.7. Resuspend pellet in 10 ml of ice cold STE Buffer.8. Add 100 ml of freshly prepared l
6、yozyme solution, incubate on ice for 15 min. Just before sonication, add 100 ml of 1 M DTT and 1.4 ml of 10% Sarkosyl. Mix thoroughly by in version and son icate for a total time of 1 mi n.9. Cen trifuge 16,000 rpm for 20 min on the SS34 rotor to pellet debris. Tran sfer super nata nt to a50-ml co n
7、ical tube and discard the pellet. Add 4 ml of 10% Triton X-100 and top up with STE Buffer to 20ml. The effective concen trati on of Sarkosyl and Trit on X-100 will be 0.7% and 2% respective ly. In cubate at room temperature for 30 min.10. Pour the lysate to 1 ml bed of prepared Glutathi one Sepharos
8、e in PBS. In cubate at room temperature for 30 min to 1 hr with agitatio n.To prepare the 50% slurry, shake up the media and pipette 2 ml to a 50 ml tube. Fill to 50 ml with PBS, in vert tube a few times. Centrifuge to 2000 rpm on a swing bucket centrifuge then switch off. Carefully suck off PBS and
9、 resuspe nd beads with 1 ml of PBS.11. Wash the beads with 3 X 50 ml of PBS. Fi nally resuspe nd in 5 ml of PBS. Pour to adispo-colu mn. Wash the 50-ml con ical tube with an additi onal 5 ml of PBS. Pool with the first 5 ml in the dispo-colu mn.To wash, use the same centrifugation technique for prep
10、aring the beads. When transferring beads to column, do not pipette but pour. The beads tend to stick to pipette tips.12. If desired, elute with 10 x 1 ml fractions of Elution Buffer. Determine desired fractions with SDSPAGE精品文档精品文档亲和层析实验技术方法INTRODUCTIONThis protocol describes a method for removi ng
11、an tibodies that react with bacterially en coded protei ns by passing a crude preparation of immunoglobulins through a column containing immobilizedbacterialprotei ns.MATERIALS* Reage ntsE. coli stra in used as host for preparati on of expressi on libraryAn tibody preparati on that is to be used for
12、 scree ningThis protocol works best when using an IgG fraction, prepared by chromatography of the antiserum on prote in A-Sepharose. Cell lysis buffer0.1 M sodium borate (pH 8.0)1 M NaClSterilize the cell lysis buffer using a 0.45-m filter, an cjk store at room temperature. Approximately 100 mlof ce
13、ll lysis buffer is required per 1 liter of bacterial culture.* Growth mediumOne liter of growth medium appropriate for the E. coli strain of choice is required.* LysozymeDissolve solid lysozyme at a concen trati on of 10 mg/ml in 10 mM Tris-Cl (pH 8.0) immediately beforeuse. Make sure that the pH of
14、 the Tris solution is 8.0 before dissolving the protein. Lysozyme will not work efficiently if the pH of the solution is less than 8.0.Use a molecular biology grade of lysozyme. Add solid lysozyme to assist lysis of bacterial cells.* NaOH (1 N)The preparation of 10 N NaOH involves a highly exothermi
15、c reaction, which can cause breakage of glass contain ers. Prepare this soluti on with extreme care in plastic beakers. To 800 ml of H2O, slowlyadd 400g of NaOH pellets, stirring continuously. As an added precaution, place the beaker on ice. Whenthe pellets have dissolved completely, adjust the volu
16、me to 1 liter with H2O. Store the solution in a plasticcontainer at room temperature. Sterilization is not necessary.* Pan creatic DNase Io 1 mg/ml Pan creatic DNase Io 50 mM NaClo 10 mM Tris-Cl (pH 7.5)o 1 mM MgCl 2Dissolve 2 mg of crude pan creatic DNase I (Sigma or equivale nt) in 1 ml of 50 mM NaCl, Tris-Cl (pH 7.5),1 mM MgCl
