Aggregation of protein after purification? - (Oct/17/2005 )
Hi, this is puzzling me for a while. After I had purified the proteins using his-tag affinity chromatography( Ni-NTA column), my elution fraction had two bands instead. One band is at the protein molecular wt, however the other band is abt twice or more larger than the prot size. Is it possible that it is dimer or aggregation? Does running SDS-PAGE disassociate possible dimer, trimer or even aggregation formation? I had done the purification under native conditions.
is the second band present in the time zero, induction and supernatant sample lanes after SDS-PAGE? it could be something that has a histidine rich area and co-purifies with your protein. Ni columns only give ~90-95% purity, if you are lucky. use other methods (size exclusion/ion exchange) to purify it even more.
it is rare that a dimer/trimer/etc. can survive the large amounts of BME/DDT that is in the loading buffer. is the band roughly 2X, 3X or 4X the MW of your protein?
Yes, I agree -- this could be just carry-over protein from the host. Have you done his-tag purification of induced host with vector only? Or, do you have western data that shows the two bands you're seeing are different forms of the same protein?