band size with flag tagged proteins? - (Apr/23/2008 )
Hi everyone, I've transfected HeLa cells with an flag-tagged expression vector. Then I carried out a western blott using a specific antibody against the overexpressed protein (no with a flag ab!!), the question is that I've obtained two different bands, one is correspondong to the native (endogenous) protein, but there is also a band with a higher size. I suppose this is the overexpressed protein, but I don't understand why does it have different size. The flag tag is too small to explain that (it's 8 aminoacids), could anyone help me, pleaz?
thanks in advance..
How much larger is it? Do you know the larger band is dependent of the flag-tagged vector? Is the smaller band running at the correct size for the native protein?
Hi, rosewater. I know the band with the lower size corresponds to the native protein because it fits with the one I see in a non-transfected extract I have done. The differences between the bands is small, just a few kDa. The native protein runs in the expected size. (approximately about 55 kDa).
thanks for answering
A few kDa is probably nothing to worry about. The tag is probably 1kDa. But I guess you'll know everything is ok when you probe with anti-flag.