Dear Matt, Based on the follow-up information that you posted, I would be willing to bet that the color you observe is caused by various Ni complexes formed with DTT/BME/etc. Dialysis won't necessarily remove these complexes (especially if protein is part of the complex) and EDTA may not destroy them either since affinity of R-S (especially if there's more than one sulfur) for Ni is typically higher than that of EDTA.
A couple of years ago I had a curious case when depending on fermentation conditions different metals were incorporated into a protein we were working with (and at that time we didn't even know it was a metalloprotein!). Metal content was low - so in dilute form all the protein preps (various mutants and truncations) were colorless, but upon concentration I was (pleasantly) surprised by a rainbow of colors - from green (it had Ni), through yellow (Mn and some Fe), into a nice coral pink (Co with traces of Fe). Some of the preps remained colorless (later turned out to have Zn). Good luck, Artem -----Original Message----- From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Matthew Alan Bratkowski Sent: Monday, September 08, 2008 12:18 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Protein Color Thanks for all of the helpful suggestions. In reply to some of the comments, I did purify by eluting with imidazole from a Ni-NTA column. The imidazole I used is 99% pure, although I'm not sure whether it is reagent or molecular biology grade. I also do use DTT in my lysis and elution buffers, so these could contribute to the color. After Ni-NTA, I dialyzed the protein into a buffer containing 10 mM Tris, pH. 7.0, 10% Glycerol, as BME overnight, and then purified it on a Q-column. I then did a final dialysis in buffer that contained 2 mM DTT and 5 mM MgCl2 (although the color appears without the MgCl2 as well), among other reagents. I would think that the two dialysis steps and the Q-column would remove all of the imidazole. However, I did not run the protein on a sizing column or use EDTA in any buffers. I could try using a sizing column, and if that does not remove the color, I could use some of the techniques suggested to determine if any metals are bound. Matt
