HIS re-purification

P.C. PC at no.email.sorry
Wed Jan 21 14:01:38 EST 2004


expression levels of your protein are not very high, I suspect 
(otherwise it would look great enough after the first pass). Have you 
checked if your protein is there after dialysis - dialysis membranes can 
adsorb an awfull lot of protein. Can it be that what you elute in SDS is 
what had not came off the first time? Did you check the flow-through 
(dialysis was sufficient, no EDTA, DTT, etc.)?

(if you used a fresh resin for the second pass and really sure that the 
amount of protein that you elute in SDS/imidazole is close to the amount 
eluted  from the first step, the above comments are irrelevant).

If it is a hydrophobic protein it can bind to sepharose in high salt 
(sepharose is a bit hydrophobic).

My experience with Ni - you need at least one or better two orthogonal 
steps to make protein really pure (depends how pure you want it to be, 
of course...). Thus, in any case, it is much "more better" to try ion 
exchange, rather than repeating Ni-chelating again.



Stephan Wenkel wrote:
> Hello,
> I have purified a HIS-tagged protein from E.coli. After dialysis against a
> buffer containing no imidazole I would like to put it again on the Ni-column
> to get rid of some minor contaminations. Unfortunately it looks like that
> the protein binds very tight to the resin that I can not get it down
> anymore. 

>Why? I can only elute it by boiling @99 degrees in the presence of
> 0,5M imidazole plus gel loading buffer.
> Thanks,
> Stephan

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