IUBio

6x his tag protein purification

Dima Klenchin klenchin at REMOVE_TO_REPLY.facstaff.wisc.edu
Sat Mar 27 16:40:00 EST 1999


In article <7dg5fs$utt$1 at news.iastate.edu>, "Dept of Microbiology" <bfeilmei at iastate.edu> wrote:
>Help!!
>
>I have cloned GFP behind a 6X his tag to purify the protein.  I am binding
>it to TALON Cobolt beads.  The protein almost binds too well, in that, I am
>having a lot of trouble eluting it off.  I have heard rumor of someone using
>histidine in the elution buffer to compete for the removal of the his tagged
>protein.  Does anyone know of a good way to elute hard to remove his tagged
>proteins?
>

It's a very good thing, really! If the strong retention has anything at all to 
do with IMAC mechanism, then, after very stringgent wash (say, 100 or
more mM imidazole) just use 100 EDTA pH 8.0 to elute everyhting -
your protein is going to be very pure! Simple dialysis will get rid of EDTA 
and cobalt. 

        - Dima



More information about the Methods mailing list

Send comments to us at biosci-help [At] net.bio.net