User:Leprevost/Purification of His-tagged proteins/Denaturing

Overview
Denaturing purifications can often lead to better purity and yield.

Lysis and column equilibration buffer

 * 8 M urea
 * 100 mM NaH2PO4
 * 10 mM Tris Cl
 * 10 mM imidazole (recommended by Kathleen, 9/27/2006)
 * pH 8.0
 * 1 mM TCEP (add just before use)

Wash buffer (Qiagen buffer C)

 * 8 M urea
 * 100 mM NaH2PO4
 * 10 mM Tris Cl
 * 20 mM imidazole (adjusted by Leprevost, 10/03/2007)
 * pH 6.3
 * 1 mM TCEP (add just before use)

Elution buffer (Qiagen buffer E)

 * 8 M urea
 * 100 mM NaH2PO4
 * 10 mM Tris Cl
 * 250 mM imidazole (adjusted by Leprevost, 10/03/2007)
 * pH 4.5
 * 1 mM TCEP (add just before use)

Grow and pellet cultures

 * 1) Grow up an overnight 5mL culture in LB plus the appropriate antibiotic.
 * 2) The following morning, dilute back the culture 1:50 to the appropriate culture volume (which depends on the expected yield of the protein).
 * 3) *Since I don't know what yield to expect, I arbitrarily did 50mL cultures assuming that my protein yield would be low and let it grow most of the day. I try to catch the cultures around OD600nm 0.6.
 * 4) Harvest the cells by centrifugation at 4000 x g for 15 mins.
 * 5) *The Qiagen protocol didn't specify a temperature so I did 4&deg;C.
 * 6) Decant supernatant.
 * 7) The cell pellet can be stored at -70&deg;C or processed immediately.
 * 8) *I stored the pellet at -80&deg;C. Long term storage at -80&deg;C might affect protein recovery???

Prepare solutions

 * 1) Prepare 10M Urea.
 * 2) Deionize the urea solution.
 * 3) Prepare lysis, wash and elution buffers (everything except reducing agent).
 * 4) Verify pH of lysis, wash and elution buffers. Adjust if necessary.
 * 5) *Dissociation of urea can lead to changes in pH. The pH definitely needs to be checked prior to using the solutions.
 * 6) Degas lysis, wash and elution buffers by placing under vacuum for one hour.
 * 7) *Tom recommends doing the degassing step last.
 * 8) Add TCEP to 1mM final concentration.
 * 9) *TCEP is not stable in phosphate buffers at neutral pH.
 * 10) Verify that pH hasn't changed.
 * 11) *It is not known whether TCEP affects pH. Degassing can affect pH in some cases.

Purify protein

 * 1) Thaw pellet for 30 mins.
 * 2) *Can thaw at room temperature since the next step happens at room temperature. However, proteases will be less active if the pellet is thawed on ice.
 * 3) *The Qiagen protocol calls for 15 mins, but it was still frozen after 15 mins so I let it thaw for 30 mins.
 * 4) Transferred to 2mL eppendorf tube.
 * 5) Resuspend in 1mL Lysis Buffer (see above).
 * 6) Add half or 1/4 protease inhibitor pellet.
 * 7) Incubate cells with agitation for 1 hr at room temperature.
 * 8) *Use an orbis shaker on the bench to do this temp (usually kept in 37&deg; incubator). Note that the shaker moves during shaking.
 * 9) Centrifuge lysate at 10000 x g for 30 mins at room temperature.
 * 10) Add 600 &mu;L lysis buffer to Ni-NTA column to equilibrate.
 * 11) Centrifuge Ni-NTA column 2 mins at 700 x g with open lid to remove equilibration buffer.
 * 12) Save 20 &mu;L cleared lysate.
 * 13) Load 600 &mu;L cleared lysate to Ni-NTA column.
 * 14) Centrifuge Ni-NTA column 5 mins at 700 x g with closed lid.
 * 15) *I typically repeat this step to load the rest of my cleared lysate.
 * 16) *Save flow through.
 * 17) Add 600 &mu;L wash buffer to Ni-NTA column.
 * 18) Centrifuge Ni-NTA column 2 mins at 700 x g with open lid.
 * 19) *Save flow through.
 * 20) Add 600 &mu;L wash buffer to Ni-NTA column.
 * 21) Centrifuge Ni-NTA column 2 mins at 700 x g with open lid.
 * 22) *Save flow through.
 * 23) Tranfer to clean 1.5mL eppendorf tube.
 * 24) Add 200&mu;L elution buffer.
 * 25) Centrifuge Ni-NTA column 2 mins at 700 x g with open lid.
 * 26) *Most of the protein should elute in this elution step.

Troubleshooting and optimization

 * Deionize urea solutions prior to use.
 * Degas all buffers.