I wish to thank everyone. I did try shifting to a higher pH and flushing 3l of
salt solution over the column which did not work. I tried 20ml 2M Urea on the
column and a stepwise shift to no urea that showed removal of the protein. I
will try binding studies to see if I did not denature the His
Depending on the type of resin you should be able to use IMAC all the way up
to pH 11 or so (works for Ni-NTA, not sure about HIS-SELECT or other
resins). Obviously your buffer system changes (carbonate works well at 11).
Why not to elute the complex together with pure monomer and then try
separat
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 p
possible (practical?) for all
proteins, but it often works very well.
Good luck,
Mike Thompson
- Original Message -
From: "Daniel Bonsor"
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 Colu
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 Nic
