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
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