Protein much larger on Western blot than predicted - (Mar/04/2009 )
Anybody who has got an idea why my protein seems to be running at around 90 kDa when it is predicted (based on the sequence) to be around 50 kDa. I am running on a normal SDS-PAGE gel (reducing and denaturing). I am pretty sure that the antibody works since knock outs lacks the band and all controls have it.
p.s. It is a nuclear receptor (RXR) it that helps with the answer ???
A (very) quick search in Pubmed retrieved a posttranslational modification (sumoylation) of RXR. There could be other too, so that may explain the shift of the band.
madrius1 on Mar 4 2009, 07:39 AM said:
Yes I looked into that, but sumoylation as far as I have read often affects only a smaller fraction of a given protein pool and I dont see any in the right size. I think there was another reason for discarding this modification, but I cant remember right now.
If you're using a pre-stained (colored) ladder to estimate MW, that could throw your results off -- these ladders are not useful for size estimation, because the migration the various bands in the ladder exhibit (the relative MW) is altered by many factors, including the pH, gel concentration, gel system and running conditions.
HomeBrew on Mar 4 2009, 08:46 AM said:
I use one prestained and one unstained....same result
are you certain you're not getting dimers?
Wrong start codon? Or, if you're working in eucaryotes, a splice variant?
aimikins on Mar 5 2009, 09:30 AM said:
Can I get that on a denaturing and reducing gel ?
HomeBrew on Mar 5 2009, 03:20 PM said:
Possibly, but these receptors are very conserved and the predicted size fits all the alignments and sizes of related and very distantly related organisms.
Thanks to everybody for suggestions, guess I might come back later with an answer if I find the solution
Post-translational modifications should not alter your target molecular weight by 40 kD and dimers would not be occuring under denaturing conditions. A splice variant would be more likely, though still such a big size difference would not be expected for receptors like RXR.
Do you have a positive control? Try to include a sample with in vitro translated RXR protein or another protein known to be ~ 50 kD.