Help! My HisTag Protein won't come off Ni Resin

Neil Saunders saunders at bio.vu.nl
Thu Feb 10 17:14:39 EST 2000


"Gareth" <gphillip at peds.bsd.uchicago.edu> wrote in message
news:38A2ED09.6922A38B at peds.bsd.uchicago.edu...
> Pamela,
>
> Could be aggregated protein that's getting left behind. Do you c'fuge/
filter
> your protein and have it in the same buffer as the column before loading?
> Or if you think the cysteines are causing a problem perhaps you could load
in
> the presence of DTT to keep them reduced.
>
> Hope that helps

But check the manual first-the concentration of DTT you can use is pretty
low, because it reacts with the nickel.  I think manufacturers generally
recommend beta-mercaptoethanol instead.

Neil Saunders

--
Department of Molecular Cell Physiology,
Faculty of Biology,
Vrije Universiteit,
De Boelelaan 1087,
1081HV, Amsterdam,
The Netherlands

tel: +31 20 4447194
fax: +31 20 4447229

email: saunders at bio.vu.nl
http://www.crosswinds.net/~nfws/index.htm










More information about the Methods mailing list