PS In fact, the protein I mentioned, was not his-tagged, and first purification step I used (after streptomycin sulphate precipitation of DNA and dialysis) was on DEAE-sepharose at pH 8. In this, the protein surprisingly bound, I would guess via bound nucleic acid contaminants, so in effect the protein may have been "chromatographing on the DNA bound to the column".
Mark J van Raaij Lab 20B Dpto de Estructura de Macromoleculas Centro Nacional de Biotecnologia - CSIC c/Darwin 3 E-28049 Madrid, Spain tel. (+34) 91 585 4616 http://www.cnb.csic.es/~mjvanraaij On 11 Jul 2013, at 09:27, Careina Edgooms wrote: > Dear ccp4 bulletin board > > Sorry for off topic question. I'm working with a protein with pI 9.5 and yet > it will not bind to a cm sepharose column when equilibrated at pH 6.5. I have > removed all salt from the buffer but it still will not bind. I wonder if > anyone has suggestions as to why this could be? Is it possible that if a > protein aggregates it won't bind? I do not suspect aggregation to be the > problem because I do filter before loading onto the column but I can't > imagine what else it could be? Prior to this step I cleave the His tag off > the protein with enterokinase over night. > > Thanks > Careina
