HIS re-purification

Stephan Wenkel wenkel at mpiz-koeln.mpg.de
Thu Jan 22 03:19:38 EST 2004


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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