What is the best way to approach (unexpected) glycosylated proteins on a western blot (or in general)?
I am looking at some potential anti-fibrotic treatments (Tx) using 2D cultured primary human myofibroblasts +/- recombinant TGF to stimulate a fibrotic phenotype, and so I wanted to look at relative abundance of the Transforming Growth Factor Receptor II (TGFBR2) protein. I just learned TGFBR2 is N-linked glycosylated, which is apparently important for transport to the cell surface and thus overall TGF-based signaling (Kim et al. J Biochem. 2012). From my preliminary blot (see photo; previously stripped blot, so a little fuzzy), it looks like my vehicle & treatment (Tx) are glycosylated (thus ~ 65-80 kDa), but the +rTGF fibrotic-model groups (including Tx before or after TGF) seem to mostly give a single band (65 kDa), so I'm guessing mostly unglycosylated.
I know I could treat my samples to remove the saccharides, get a single band and quantify for relative abundance, but wouldn't that be removing some potentially important information? Before I waste a lot of time and money, I'm wondering what is the smartest way to approach this? Based on qPCR my Tx doesn't change gene expression of TGFBR2, so I'm not surprised if I don't see differences in TGFBR2 protein volume, but I thought this glycosylation thing might be interesting. I appreciate everyone's time and thoughts on this matter.
You should certainly treat with PNGase F to learn if the difference is in fact due to glycosylation. That's your hypothesis. You need to test it, before even thinking about how to study glycosylation.
If it's actually true -- meaning your result reproduces at all, and then all the higher bands disappear under the influence of the deglycosylating enzyme -- then I agree, that would be really interesting. You would best study this further by separating the released glycans and analyzing them by mass spectrometry. Also chop the protein up with trypsin and study the released peptides with glycosylation still on them. It's probably most efficiently done through a full service contract lab, if you don't have that expertise and instrumentation in your group.
If you just want to measure relative abundance based on Western blot band quantitation, then you should be able to add together the densities of the glycosylated and unglycosylated bands. It would be wise to also check once that you get the same result if you enzymatically deglycosylate, just in case the signal is different for glycosylated and unglycosylated proteins.
The coomassie brilliant blue stained gels may be subsequently stained for glycogen moiety with PAS (periodic acid Schiff) staining.
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