Hello Dan,
another denaturing option worth trying is to do it on the nickel column.
Purify via your normal means to start but then while your protein of interest
is bound to the nickel resin (with the sticky partner bound to it), you can
wash with buffer containing increasing amounts of urea (or Gd-HCl) and then
continue to wash while slowly decreasing the amount of denaturant. Finish off
with a very thorough wash in your original denaturant-free wash buffer and then
elute. Your protein of interest should remain bound to the Ni column
throughout but the sticky protein should be washed away and (if you are lucky)
your protein will then refold while bound to the column and you will elute
native protein minus your troublemaker. You may not even have to go all the
way up to 8M urea or 6M GdHCl, only using a small amount of denaturant may be
sufficient to remove the troublemaker and your protein of interest may not be
completely denatured in the process making refolding less of a proble!
m.
good luck,
Eric
__________________________
Eric Larson, PhD
Biomolecular Structure Center
Department of Biochemistry
Box 357742
University of Washington
Seattle, WA 98195
On Wed, 17 Nov 2010, Michael Thompson wrote:
| 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
|
