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
> >>>
> >>>
> >>
> >
> >
>
