p16 antibody in western blotting, weird results - western blotting, p16 (Mar/02/2005 )
Welcome everybody, hello, nice to meet you
And I have a question to start with
I am getting very strange results with the p16 antibody on my western blots. The stripes seem to be completely unspecific, they migrate higer than they should, they do not differ (I should have differences between my cell lines). More, there shouldn't be many stripes - it's a mouse monoclonal Sigma antibody.
Has anyone used it? Or another antibody against p16? This one is against 12 c-terminal aminoacids.
Is checking mRNA expression a good idea? Are other antibodies better? I can't honestly risk immunoprecipitation with the results I am getting, that makes no sense.
I will be very grateful for any suggestions.
in order to reduce the unspecific bands, you can block your membrane 2h at room temperature (TBS/PBS 5% fatless powder milk).
you can let hybridize your primary anticorps overnight at 4°C, and the second one the night after.
did you contact sigma in order to ask them if the p16 antibody recognizes several proteins and the appropriate dilution to work with? You can also contact them, and they could help you.
I had no multiple unspecific bands at lower concentration (1:200), but my stripe was showing a protein approx 80 kDA. And there were no differences between the cell lines, too. I would like to have any specific bands, but there's nothing at 16 kDa. I've verified the blot with Ponceau Red, I have some proteins there, so...
The data sheet says it should be 1:1000, and with this dilution I have no bands at all. Maybe I should contact Sigma.
So...have anyone tried a different one? Or worked with this one? That would be good
Do you have a positive control? p16 is often deleted in cell lines and may not be expressed in your cells. In primary cells p16 expression will be negative if the cells are in cycle. If you can get hold of some HeLa cells they make a great positive control as they massively overexpress p16 at all times. Many other lines do as well.
I just figured it out in the meantime Though my cells should express p16, they are genetically unstable and they could just lose the expression.
I am trying to get the HeLa cells now. Anyway, thank you I'll post to tell if it works