Hola Careina, filtering won't help much against aggregation, it just removes the larger aggregates, but the protein may still form small aggregates that pass the filter.
and remember the pI from the sequence is only an estimation and may or may not be close to the real pI, which can only be measured by iso-electric focussing. A protein I worked on in my thesis had a theoretical pI of over 9, but a real pI of 7.5 or so. This protein forms specific oligomers, perhaps screening positively charged groups from solution. another possibility is that your protein binds nucleic acids or other charged contaminants from your expression host and that those screen the charge from solution. 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
