>> We are studying a human protein that we suspect has a tendency to aggregate in
> vivo. After size exclusion chromatography and Western blot analysis of the
> fractions with polyclonal antibodies,there are two strong signals: one in the
> first fraction, and one in later fraction. Presumably the first fraction
> contains an aggregated form of the protein. My question is: how do we show
> that the protein present in both fractions is the same? We do not want to rely
> on the antibodies only, and we cannot do microsequencing, the amounts are too
> low. Limited proteolysis is not agood idea either if it is the same protein in
> different conformations. Any suggestions, please?
2D electrophoresis. The probability that two different proteins have the
same subunit MW, the same pI and react with the same antibody should be
very low indeed.