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
>>
