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Sandy, like mentioned previously, sounds like FeS. record an optical spectrum. Or even better, check whether there is somebody on the campus running an EPR machine (equipped for helium temperature measurements) Maybe a new FeS protein? FeS is not necessarily required as redox cofactor. It can have any other function . See e.g. primase from eukaryotes (recent structures) Or in your native (maybe eukarytic) protein is a Zn. E.coli places sometimes a FeS instead of a Zn in the site. Less likely but further possibility: Ni2+ bound to the protein. If DTT is added: Ni2+ -S coordinated is not redox stable and oxidizes to Ni3+ (brownish colour). HTH Guenter > Dear all, > > I have purified protein from E.coli. expression system. the protein > has been purified with three independant columns. Now during > concentration step using amicon, the protein shows brown colour. what > could be the reason. > > best regards and Thanks, > sandy > <http://sigads.rediff.com/RealMedia/ads/click_nx.ads/www.rediffmail.com/signatureline.htm@Middle?> > >