Editing Protein Purification from Inclusion Bodies Protocol (section)

== Purification of inclusion bodies<br> ==

<br>Stock solutions<br>â¢ lysozym 50 mg/ml in dH2O (store at -20 C)<br>â¢ DNase 1 mg/ml in 50% glycerol, 75 mM NaCl (store at -20 oC<br>â¢ MgCl2 0.5 M in dH20<br>â¢ Tris-Cl 2.5 M pH 8.0<br>â¢ NaEDTA 0.5 M in 50 mM Tris-Cl, pH 8.0<br>â¢ NaAzide 30% in PBS<br>â¢ Na deoxycholate 10% in dH2O<br>â¢ Triton X-100 10%<br>â¢ 5 M NaCl<br>â¢ DTT 1 M

'''Buffers'''<br>

<br>'''Solution buffer''' (13 ml), pH 8.0<br>50 mM Tris-Cl 260 ÎŒl stock<br>25% sucrose 3.25 g<br>1 mM NaEDTA 26 ÎŒl stock<br>0.1% NaAzide 43.3 ÎŒl stock<br>10 mM DTT 130 ÎŒl stock

'''Lysis buffer''' (12.5 ml)<br>50 mM Tris-Cl 250 ÎŒl stock, pH 8.0<br> 1% Triton X-100 1.25 ml stock<br> 1% Na deoxycholate 1.25 ml stock<br> 100 mM NaCl 250 ÎŒl stock<br> 0.1% NaAzide 42 ÎŒl stock<br> 10 mM DTT 125 ÎŒl stock

'''Washing buffer with Triton''' (10 ml)<br>50 mM Tris-Cl 200 ÎŒl stock<br>pH 8.0 0.5% Triton X-100 0.5 ml stock<br>100 mM NaCl 200 ÎŒl stock<br>1 mM NaEDTA 20 ÎŒl stock<br>0.1% NaAzide 33.3 ÎŒl stock<br>1 mM DTT 10 ÎŒl stock<br>

<br>'''Washing buffer without Triton''' (10 ml)<br>50 mM Tris-Cl 200 ÎŒl stock<br>pH 8.0 100 mM NaCl 200 ÎŒl stock<br>1 mM NaEDTA 20 ÎŒl stock<br>0.1% NaAzide 33.3 ÎŒl stock<br>1 mM DTT 10 ÎŒl stock

11. Resuspend pellet in 13 ml solution buffer on ice and transfer it to a 30 ml centrifugion bottle<br>12. Sonicate 50% level 4-5, 30 pulses, on ice<br>13. Add 100 ÎŒl lysozym, 250 ÎŒl DNase I, 50 ÎŒl MgCl2, vortex<br>14. Add 12.5 ml lysis buffer and vortex short<br>15. Incubate 30-60â² at RT<br>16. Add 350 ÎŒl NaEDTA<br>17. Freeze in N2 until it stops bubbeling and thaw for 30â² at 37 oC<br>18. Add 200 ÎŒl MgCl2<br>19. Wait until viscosity decreases 30-60â²<br>20. Add 350 ml NaEDTA

From now on everything should be done on ice<br><br>

21.&nbsp; Pellet at 11,000 g, 20â², 4 oC<br>22.&nbsp; Discard supernatant and take a sample<br>23. Resuspent pellet in 10 ml washing buffer with Triton<br>24.&nbsp; Sonicate 50% level 4-5, 30 pulses, on ice<br>25. Pellet at 11,000 g, 20â², 4 oC<br>26. Discard supernatant and take a sample<br>27. Resuspent pellet in 10 ml washing buffer without Triton<br>28. Sonicate 50 % level 4-5, 30 pulses, on ice<br>29. Pellet at 11,000 g, 20â², 4 oC<br>30. Discard supernatant and take a sample