I used some polyclonal western blot antibody, and that datasheet size is 45-50kDa
I also get the band in expected size range, but there's multiple other band(at least 3 bands in other size), and when i found the references about that protein, there's various size (e.g. 35kDa, 63kDa, 75kDa)
In this case, how can I confirm that 45-50kDa band is really my target protein?
Can I use the protein sequencing (service that supplied by company using SDS-PAGE band, N-terminal partial amino acid sequencing) for check that 45-50kDa band is identical with my target protein amino acid sequence?
Is this protein sequencing can apply for target band validation?
Mass spectrometry of protease digests of bands cut out of SDS-PAGE can be used to identify the protein, if it is sufficiently separated from other proteins. If you are running a crude extract instead of a purified or immunoprecipitated protein, there will probably be several proteins in the band.
@adam B Shapiro is it okay if he first does immunoprecipitation assay to pull down his protein of interested inorder to establish the exact location of the band of interest? Then the next time he is dealing with the crude extract he doesn't have to worry about other bands.
Bbumba Patrick I agree that immunoprecipitation would be worthwhile. If the antiserum is specific enough, it should result in an SDS-PAGE profile that is much simpler than the crude extract, making it easier to obtain useful information from the mass spec experiment.
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