and possibly the opposite to reduction which is oxidation - do you have cys residues? Perhaps your DTT or TCEP got exhausted? Remember you can add up to 10mM (not the traditional token 1mM). But remember to neutralize TCEP. Other possibility is adventitious metals as these give strong charge transfer bands on binding to cys.
So make sure you include EDTA or similar chelator (after metal affinity obviously). Oxidation is more likely if your protein unfolds - so try to keep it happy with nicely buffered pH and any ligands that might stabilize it. Good luck. Martyn Martyn Symmons Cambridge --- On Fri, 24/9/10, vikrant saa <[email protected]> wrote: From: vikrant saa <[email protected]> Subject: Re: [ccp4bb] protein turns brown To: [email protected] Date: Friday, 24 September, 2010, 10:50 sometime it does happen becoz of protein aggregation, or reducing environment. but it may be your protein color as well that visible during concentration Vikrant From: sandeep <[email protected]> To: [email protected] Sent: Fri, 24 September, 2010 2:04:54 PM Subject: [ccp4bb] protein turns brown 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
