Dan, You could try a denaturing purification to get rid of the binding partner.
First, take your cell lysate and spin it down at high speed to remove insoluble contaminants. (You probably do this already.) Then take your clarified lysate and dialyze it into buffer containing 6M Guanidine HCl. This will unfold everything, including proteases (no proteolysis under these conditions), and break the interaction between your protein and its binding partner. Then you can spin again at high speed to remove any additional aggregates and load this denatured sample onto the nickel column. The His-tag will still stick if the protein is unfolded in the presence of 6M GdHCl. Elute the protein in the same buffer with 6M GdHCl plus high imidazole concentration. Then you can take your eluent and dialyze out the GdHCl to refold the protein. Refolding (in the test tube, anyway) is not possible (practical?) for all proteins, but it often works very well. Good luck, Mike Thompson ----- Original Message ----- From: "Daniel Bonsor" <bon...@bbri.org> To: CCP4BB@JISCMAIL.AC.UK Sent: Wednesday, November 17, 2010 8:49:37 AM GMT -08:00 US/Canada Pacific Subject: [ccp4bb] Off Topic - Nickel Column I have a His-tagged protein which I am coexpressing with it's binding partner to prevent proteolysis. Once on the Nickel column I can remove 80% of the partner by flushing 2l of 1.3M NaCl solution buffered at pH 8.5 overnight. However the last 20% is difficult to remove, even if I reload the Nickel column and flush a further 2l of salt solution. I am wondering if I can increase the pH to 9.0 or 9.5. It should not effect the binding of His for the Nickel as the His-tag has to be deprotonated to bind, though will it causing stripping of the Nickel? Thanks Dan -- Michael C. Thompson Graduate Student Biochemistry & Molecular Biology Division Department of Chemistry & Biochemistry University of California, Los Angeles mi...@chem.ucla.edu
