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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 * >> >>