HIS TAG and antibody affinity purification

Tom Thatcher ttha at troi.cc.rochester.edu
Tue Jun 7 20:27:41 EST 1994


In <01HD9IEJFCW2002C7F at msvax.mssm.edu> GLUXLAB at MSVAX.MSSM.EDU (STRUCTURAL NEUROBIOLOGY XTAL LAB- MADE FROM 100% RECYCLED ELECTRONS) writes:


>Dear Methodists and Reagentites-

>	With all of the vast resources and
>experience out there in 'net'land, has anyone
>come up with a way to cross link a protein
>coupled with a HIS tag to a nickel [or
>other column] for the purpose of affinity 
>purifying antibodies. 

>	I do not want to activate CNBr-Sephadex
>but exploit the property of a rapid purification 
>on one column. I am hoping that the interaction
>between the His Tag and the nickel-NTA column
>is stronger than the antigen-antibody interaction.

I'm afraid just the opposite is true.  The two proteins I purified
with a his tag eluted at around 100 mM salt--far lower than typical
antibody-antigen binding affinities (we do Westerns in up to
400 mM salt to reduce non-specific binding with one of our Abs.)

Tom Thatcher
University of Rochester Cancer Center
ttha at troi.cc.rochester.edu



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