Hi, Common issue indeed (a score of E. coli proteins have high affinity to IMAC media). I've never seen this particular protein as a contaminant, but then again I do not always identify all the contaminants as long as I can get rid of them :)
Things to try: 0. if you really suspect a specific interaction with your protein - then the thing to check is a 'blank' E. coli extract loaded and eluted in the same conditions. Does the offending contaminant not bind in the absence of your target protein ? If you discover that the interaction is specific then you have to experiment - salts, detergents, polyols, NDSB's - this kind of stuff can help you dissociate the two. Even low concentrations of urea or guanidine hydrochloride - anything goes at that stage. 1. different media and metal - Ni-NTA, Co-NTA, His-SELECT (Ni), His-SELECT (Co), TALON, HisTrap, etc. Depending on the resin you would need to elute with varying amounts of imidazole so your best bet is to always elute with a gradient at first. 2. As suggested by others - HIC is a very useful and frequently neglected method of separation. 3. Good old fashined AS cut. 4. A glucosamine column or something similar (heparin column?) - it may suck out the contaminant based on affinity. 5. Cleaveable His-tag, as suggested earlier. 6. treat the protein mixture with one of the native ligands (just like ATP and GroEL/S Artem -----Original Message----- From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Ailong Ke Sent: Friday, November 02, 2007 2:43 PM To: [email protected] Subject: [ccp4bb] Ni-NTA purifications contaminated with E coli Glucosamine-fructose-6-phosphate aminotransferase >Hello, We are trying to purify an N-terminal His6-tagged protein from E. coli, and the prep was contaminated with the E coli Glucosamine-fructose-6-phosphate aminotransferase, which co-purifies with my protein of interest in subsequent ion-exchange and sizing columns. This protein appears to be a common contamination in the Ni-NTA purifications. Does anyone have tricks to get rid of it? Thanks. Ailong --
