>I am trying to bind a 180 KDa protein with a His tag to a nickle column
>(invitrogen) but most of the protein comes out in the flow through. I
>have tried both native and denaturing conditions. Can anybody offer any
>suggestions?
Have you checked your buffering conditions during binding to make sure they
are not reducing the Ni++? Make sure the pH is >7, and make sure you have
little reducing agent (<1mM DTT, <20mMM BME).
_______________________________________
Antonin Tutter
Salk Institute for Biological Studies
RBIO-J
10010 N. Torrey Pines Rd.
La Jolla, CA 92037
email: atutter at aim.salk.edu
web: http://www-biology.ucsd.edu/~atutter/
