native HisTag elution alternative

Michael L. Sullivan mlsulliv at facstaff.wisc.edu
Fri Oct 27 08:46:18 EST 2000


>
>We now want to turn to an alternative elution
>approach e.g. EDTA. Has someone made good ex-
>perience with this under native conditions and
>could provide us with a protocol?
>

Several vendors suggest 100 mM EDTA pH 8.0 as an EDTA elution condition.  I
had tried this with my his-tagged protein, but in my case the protein
seemed to bind very well to the agarose support itself and so only ever
came off the matrix by boiling in SDS-PAGE sample buffer (this was despite
denaturing conditions and high NaCl!).  In theory, however, EDTA should be
very effective at eluting off your protein, and in fact I would think would
do so at a concentration well below 100 mM.

I have a question now though.  Has anybody tried eluting proteins with
Co2+, Ni2+, or Cu2+?

Mike

Michael L. Sullivan, Ph.D

U.S. Dairy Forage Research Center
1925 Linden Drive West
Madison WI, 53706

(608) 264-5144 Phone
(608) 264)-5147 Fax


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