Protein Size 50 kDa higher than expected
Posted 07 September 2012 - 09:51 AM
Our protein of interest is expected to be ~95 kDa, reported by multiple antibody providers, Ensembl, Uniprot, etc. Bizarrely, some of these antibody providers state the expected size is 95 right next to a gel image that shows it at 130. Others show a gel image with bands at both sizes. I've contacted 3 of them about this, 1 didn't answer and the other two took a week to admit they didn't have any explanation.
With two different antibodies in multiple blots, we see a strong band at ~150 and either nothing or a much weaker band at 95. We also have a commercial positive control lysate from a cell line transfected to overexpress the protein. In multiple blots with the two antibodies, it has bands at both sizes.
I'd chalk it up to cross-reactivity and assume the 95 is correct, except that I also did a transient siRNA knockdown. Comparing the target knockdown sample and the scramble knockdown sample, the 95 and 150 bands are present in both samples and both are markedly weaker in the target knockdown. Sample loading was even by B-actin. The siRNA knockdown was also tested by qPCR at the same timepoint and found to be very specific.
So that suggests both bands are in fact the target.
-There are multiple isoforms of the protein, but they are all nearly the same size (~95-100 kDa).
-None of the post-translational modifications I know of would account for a 55 kDa discrepancy except for possibly glycosylation- but there's no glycosylation of this protein on record, and both bands are tight on the blot with no sign of smearing.
My other thought is that there's a strong protein-protein interaction or some folding making it through the denaturation step, but I've tried pretty stringent conditions:
4x LDS NUPAGE loading buffer from Invitrogen + 0.45 ul B-mercaptoethanol ea sample
NuPAGE running and transfer buffers
4-12% Bis-tris 10 well gel
95º C boiling (5, 10, 15 minutes)
I'm kind of stumped at this point. What am I missing?
Posted 07 September 2012 - 10:36 AM
Posted 10 September 2012 - 04:54 AM
Posted 10 September 2012 - 11:40 AM