I am trying to run a western blot on catechol-o-methyltransferase (COMT) in the rat brain. All of my other proteins of interest (TH, GAPDH, Vinculin) are showing up exactly where they're meant to be; however, COMT is not. It is meant to be ~30kDA, but it's showing up at ~60. I was concerned that perhaps it was not fully denatured, so I have tried increasing my ß-ME from 10% to 20%, as well as tried heating it for 10 minutes at 95C instead of 5 minutes (which works for everything else I have). Neither had any effect. I have replaced the antibody, but the company I got it from sent me more from the same batch for whatever reason. Is it worth it to try adding DTT, or what are my next steps? I do not have access to mass spec to confirm if what's being labeled is COMT or not, unfortunately.
Your reduction conditions are sufficiently aggressive already. The most likely explanation is that you have a poor antibody. Does the supplier actually show any western data for rat brain? ideally, you need some pure comt reference protein. One possibility I suppose is that 60k band is a spurious signal far stronger than the one you expect at 30K. Does a longer exposure reveal a 30K band?
I have considered it might be dimerising, but I would expect that the added ß-ME and heating time would give me at least SOME of my protein at the target KD if that were the case. I have exposed my gels from 30 seconds all the way up to 8 minutes, and there is no band at the correct weight.
Thank you very much Nick Gee and James Matthew Minshull for your replies! I have just received a replacement Ab from the vendor that is a mouse monoclonal (not the goat poly I had) and I am hopeful it will work -- I just wanted to make sure I was not missing an obvious step that could fix things.
Do you know if those protein is suppose to be highly glycosiolated?
Since the hteoreticall MW do not consider this kind of PMT that in some cases may increase a lot the MW of your protein. In the past i worked with an high glycosilated proteins which has a theoretical MW of 30KDa but in the gel run as 90KDa.
In case that you have many predicted glicosilation sites, YOu can try to treat your sample with PNGases, ENdoH or other de.glycosilating enzimes and see if it reduce the observed MW.
YOu can predict theoreticall glycosilation sites with online web servers as
https://services.healthtech.dtu.dk/service.php?NetNGlyc-1.0
https://services.healthtech.dtu.dk/service.php?NetCGlyc-1.0
https://services.healthtech.dtu.dk/service.php?NetOGlyc-4.0
good luck
Manuele
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