Editing RNase A Purification

1. Grow BL21 to log phase by shaking at 37°C.<br>2. Add IPTG 0.5 mM and induce for 4 h to o/n.<br>3. Spin down cells.<br>4. Freeze cells.<br>5. Resuspend in cell lysis buffer, 20 mM Tris-HCl buffer, pH 7.8, 6M urea, 1 mM EDTA.<br>6. Shake 20 min at 37°C. <br>7. Spin for 15 min at 30,000 xg.<br>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<br>9. Shake for 20 min at 37°C. <br>10. Spin for 15 min at 30000 g.<br>11. Add reduced DIT 0.22 g to the resulting supernatant<br>12. Stir the solution at room temperature for 10 min.<br>13. Add Glacial acetic acid to adjust the pH to 5.0.<br>14. Dialyze exhaustively against 20 mM Tris-acetic acid (AcOH) buffer, pH 5.0, 100 mM NaCl<br>15. Remove insoluble protein by centrifugation. <br>16. Collect the soluble fraction and reoxidize by exhaustive dialysis (&gt;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).<br>17. Dialyze refolded protein vs 20 mM <strike>Tris-AcOH</strike> Tris-HCl buffer, pH 8.0.<br>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. <br>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.<br>20. Wash the column with the same buffer (100 ml). <br>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.<br> 

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

[[Category:Protocols]]