I ran Western Blot and didn't see the bands for my protein of interest in the right place, however, there are other bands that are 10 KD above the expected, there might be post translation modification but I am not sure since it is a new molecule and little is known about it. how can I know that those are the bands I am looking for or not?
Thanks
Membrane proteins never run with the exact pattern like the ladder. Which ladder did you use which is giving you a 10 KDa separation ? The only thing which you may do is treat your sample in such a way that your glycosylation will be totally removed and load it on the gel next to the untreated sample and then compare
A few suggestions:
- per above, yes the bands are rarely going to run exactly like the ladder, so the ladder is only an estimate of molecular weight. This is especially true at the lower end of any run - you mention 10kDa, so that might be near the 'bottom' of the gel. You could try running a higher crosslinked gel to better resolve this smaller target protein.
- related possibility is that your protein might be structured as a dimer or in a complex with other proteins. Typically SDS denatures such structures, but you didn't actually mention that you're using SDS. You might try aggressive denaturation (boiling) before loading the gel.
- cut out the band and do typical bottom-up proteomics analysis: tryptic digestion followed by MS (or LC/MS) to identify the peptides. This I think would be a preferred method to prove the protein identity since the peptide information is truly 'orthogonal' (biochemically independent) information to the antibody binding from your Western.
- also mentioned above, strip off any possible glycosylation (enzymes or
trifluoromethanesulphonic acid) AND you should also treat with a phosphorylase in case there might be phosphate groups. Be aware that such treatments are not always quantitative (especially when you are working on a 'new' protein).
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