Paul.xu at 14:30, 7 May 2014

2014-05-07T14:30:18Z

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1. Grow BL21 to log phase by shaking at 37°C. 2. Add IPTG 0.5 mM and induce for 4 h to o/n. 3. Spin down cells. 4. Freeze cells. 5. Resuspend in cell lysis buffer, 20 mM Tris-HCl buffer, pH 7.8, 6M urea, 1 mM EDTA. 6. Shake 20 min at 37°C. 7. Spin for 15 min at 30,000 xg. 8. Resuspend the pellet in 250 ml of solubilization buffer, 20 mM Tris-HCI buffer, pH 7.8, 6M urea, 0.4M NaCl, 20 mM DTT, 1 mM EDTA 9. Shake for 20 min at 37°C. 10. Spin for 15 min at 30000 g. 11. Add reduced DIT 0.22 g to the resulting supernatant 12. Stir the solution at room temperature for 10 min. 13. Add Glacial acetic acid to adjust the pH to 5.0. 14. Dialyze exhaustively against 20 mM Tris-acetic acid (AcOH) buffer, pH 5.0, 100 mM NaCl 15. Remove insoluble protein by centrifugation. 16. Collect the soluble fraction and reoxidize by exhaustive dialysis (>24 h) against refolding buffer, 50 mM Tris-AcOH buffer, pH 7.8, 0.1 M NaCl, 1 mM reduced glutathione 0.2 M oxidized glutathione (0.2 mM). 17. Dialyze refolded protein vs 20 mM <strike>Tris-AcOH</strike> Tris-HCl buffer, pH 8.0. 18. Load sample to a column <strike>(15.0 cm X 5 cm2 )</strike> 5 ml of <strike>DE52 anion </strike>exchange resin Q FL equilibrated with the same dialysis buffer. 19. Collect flow-through, and then load to a column <strike>(15.0 cm X 1.8 cm2)</strike> of <strike>S-Sepharose</strike> SP cation exchange resin equilibrated with <strike>Tris-AcOH</strike> Tris-HCl buffer (20 mM), pH 8.0. 20. Wash the column with the same buffer (100 ml). 21. Elute RNase A with a linear gradient of NaCl (0.00-0.35 M), in <strike>Tris-AcOH</strike> Tris-HCl buffer, pH 8.0. 

22. Collect 5ml fractions and analyse purity by SDS-PAGE.

[[Category:Protocols]]

RNase A Purification - Revision history

Paul.xu at 14:30, 7 May 2014