IUBio

HIS re-purification

P.C. PC at no.email.sorry
Thu Jan 22 11:48:19 EST 2004


how about cysteins in your protein?
The problem could also be oxydation of the purified protein (during 
dialysis, or on the column).

does it normally form disulfide-bonded multimers? (in this case you can 
try to reduce pH to keep Cys native).

all shoting in the dark, of course.
good luck,


Peter

Stephan Wenkel wrote:

> I tried batch purification and because I was desperate I also tried another
> protein I purified earlier, same result.
> what else?
> 
> "P.C." <PC at no.email.sorry> wrote in message
> news:buoo8n$qjk$1 at cruncher.dfci.harvard.edu...
> 
>>aggregation? -try to bind "in batch" (?)
>>
>>Stephan Wenkel wrote:
>>
>>>Hello,
>>>
>>>there is a lot protein present. I checked on gel and on western.
>>>If I put it a second time on the resin I can not elute it anymore only
> 
> with
> 
>>>very harsh conditions.
>>>Any other suggestions appreciated.
>>>Regards,
>>>Stephan
>>>
>>>
>>>"P.C." <PC at no.email.sorry> wrote in message
>>>news:bumict$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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