Imidazole and complex stability

[Bean Long]

DK wrote:
> 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
> 
> DK

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?

-- 
Bean

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