Imidazole and complex stability
ben.long at yourfinger.anu.edu.au
Thu Jul 13 21:32:21 EST 2006
> In article <44b6e1cc$1 at clarion.carno.net.au>, ben.long at yourfinger.anu.edu.au wrote:
>> Hi all,
>> I have several complexes which I have purified via a 6xHis tag on one of
>> the components. I elute from IMAC with 250 mM imidazole and can show,
>> using SDS-PAGE, that the tagged protein and several other complex
>> components have co-eluted. Controls confirm that these co-eluting
>> components are not non-specifically bound to IMAC. When the purified
>> complex is run on native PAGE, however, the tagged protein doesn't run
>> as a complex with the other proteins. I was wondering if the high
>> concentration of imidazole might be pulling the complex apart?? I am
>> going to try cross-linking prior to IMAC but wondered if anyone had
>> heard of potential complex dissociation due to high imidazole before??
> If it were high imidazole concentration dissociating the complex,
> you'd have complext forming in the dialysis bag when you dialyse
> imidazole out. Di you do this trivial experiment?
> Also, it could be that native electrophoresis conditions compromise
Thanks DK. I've actually been concentrating and doing buffer exchange
with a MW cutoff filter but this may be too harsh. I'll give dialysis a
go and see what I end up with. A potential problem has been the
requirement to concentrate.
Electrophoresis conditions may be causing some dissociation too. Any
recommendations on available buffer systems which may be milder?
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