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Hi Fred, I doubt His-trap column be any better. I never found much a difference between these resins or columns. If you want to try, use His-Trap HP, not His-Trap FF. Doing batch binding may help you figure out the problem. In some cases, protein binds better in batch mode. You can take beads out at time intervals up to overnight for binding analysis. I guess not working means the protein in the flow-through. Sometimes for native purification, proteins don't come out of a column. But never experienced anything like this under denatured conditions. Good luck, Chun -----Original Message----- From: CCP4 bulletin board [mailto:[log in to unmask]] On Behalf Of Fred Sent: Wednesday, January 28, 2009 12:08 PM To: [log in to unmask] Subject: Re: [ccp4bb] [OFF TOPIC] his-tag doesn't bind Just to let you know. No way, Talon also don't work. I am gonna try the GE His-trap column. Fred wrote: > Hi everyone, > Thanks for answer my question. Just to add some more notes regarding to my expression system. The insert-vector (pET28) has been sequenced and the his-tag is N-terminal. The anti his-tag WB is positive and the binding buffer's pH is 8.2 (double-checked). > I had already experienced the same problem before, which I solved just increasing urea from 6M to 8M. Now, I have reached GndHCl 6M and no binding at all. > I'm currently running a SDS-PAGE with samples eluted from Talon and let you know the results. > All the Best, > Fred >> >> >> --- Fred /<[log in to unmask]>/ schrieb am *Di, 27.1.2009: >> * >> >> *Von: Fred <[log in to unmask]> >> Betreff: [ccp4bb] [OFF TOPIC] his-tag doesn't bind >> An: [log in to unmask] >> Datum: Dienstag, 27. Januar 2009, 22:00 >> >> * >> >> *Hi ccp4 list, >> I am trying to purify a his-tag protein by metal affinity chromatography. The >> protein was expressed in inclusion bodies and its his-tag doesn't bind the >> Qiagen Ni resin in denatured conditions (urea 8M or GndHCl 6M). Playing with >> NaCl and detergents didn't help much. >> Any help is appreciated. >> Fred * >> >>