HIS TAG and antibody affinity purification

Marko Hyvonen hyvonen at embl-heidelberg.de
Fri Jun 10 04:54:01 EST 1994


>>	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

I would have few comments on both articles above. 
First I think Tom Thatcher, with all do respect, has mixed up things a bit. Or
then we are talking of different things. Interaction of his-tagged (six
histidines usually) protein with Ni2+-NTA agarose is very strong and it is not 
affected by salt, even 2M NaCl. Protein stays bound to the column also in 6M
GndHCl and 8M urea. Protein is however eluted with 100 mM imidazole due to
competition of binding to Ni2+. 
Perhaps Tom Thatcher meant 100 imidazole when he said salt. If his-tagged
protein is eluted with 100mM NaCl, I would claim that protein was bound to the
column by ionic interactions, not by interaction of his-tag with nickel ions.
To the original posting on antibody purifications with his-tag immobilized
antigens, I would say that it should work. The problem could be the elution
conditions required. You can use high salt, urea and guanidinium (pH close to
7), but you cannot use low pH commonly used in antibody purifications, since
this will also elute your antigen from the column. 
I have never used my his-tagged proteins for antibody purifications, but there
was an article in Methods: A Companion to Methods in Enzymology (1992) 4:73-78
where authors describe a method for immobilizing his-tagged proteins to
Chelating Sepharose through kinetically inert Co(III) linkage. This provides
very high affinity. Briefly, protein is bound to Co(II)-Chelating-Sepharose in
the absence of oxygen and then Co(II) is oxidized with atmospheric oxygen to
Co(III). Co(III) is unable to change ligands and resulting matrix can be used
for antibody purification (shown in the article). This immobilization protocol
allows also acid elution of antibodies from the column. 
I have never tried this, but it sounds good.


    					regards, Marko Hyvönen
                                        hyvonen at embl-heidelberg.de






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