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
