help! binding to nickle column

Antonin Tutter atutter at
Fri Aug 14 02:54:00 EST 1998

>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

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
10010 N. Torrey Pines Rd.
La Jolla, CA  92037
email:  atutter at

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