Different mol wt band compared to bioinformatic prediction algorithms - (Jan/06/2010 )
Its very strange. When I feed in the amino acid seq of the protein of my interest into protein properties prediction algorithms (say protparam), I get 88kDa as predicted mol wt. But when I do a western, I see a band at 120kDa. The protein is not much glycosylated, not a multimer, and its a membrane anchored protein. Its not the antibody because we got this antibody from hybridoma bank (a paper has been published with that, and I cannot email those authors because they are my competitors). Any ideas?
Thanks a lot!
what do you mean by "not much glycosylated"?
the difference between predicted and actual molecular weight is usually caused by post-translational modifications.
the fact that the protein anchors to a membrane would indicate such modifications.
also, keep in mind that molecular weight determination by sds-page is not exact. there are a number of factors that can effect the apparent molecular weight.
if you want to get a better determination of molecular weight then you should use a mass spectrometer.
Thanks mdfenko. When I used glycosylation prediction programs, it said that there are just 3 sites possible, and the literature does not cite this molecule as heavily glycosylated. Even if they are, a difference of 30KDa is too much isnt it? Do membrane proteins give higher apparent mol wt in SDS-PAGE gels?
But your point on mol wt determination by SDS-PAGE is true.
3 sites for attachment does not necessarily mean that only three molecules of sugar will be bound. glycosylations are often tree structures so they can impart significant increase in weight and size.
also, glycosylation can effect binding of sds thus altering mobility of the protein.
there is no general rule about apparent molecular weight of membrane proteins.
And, don't use a pre-stained ladder if you're trying to be accurate about MWr...