IUBio

HIS re-purification

P.C. PC at no.email.sorry
Thu Jan 22 09:54:06 EST 2004


aggregation? -try to bind "in batch" (?)

Stephan Wenkel wrote:
> Hello,
> 
> there is a lot protein present. I checked on gel and on western.
> If I put it a second time on the resin I can not elute it anymore only with
> very harsh conditions.
> Any other suggestions appreciated.
> Regards,
> Stephan
> 
> 
> "P.C." <PC at no.email.sorry> wrote in message
> news:bumict$p2o$1 at cruncher.dfci.harvard.edu...
> 
>>interesting,
>>
>>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.
>>
>>best,
>>
>>Peter
>>
>>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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