Hi, i´m looking for an anti-FLAG antibody. I have used the antibody from Cell Signaling, but it seems to have very weak signal in western blot. I have loaded 75ug of nuclear protein (since the flag coupled protein that I want to detect must be only in the nucleus), 1:500 of primary antibody and 1:1000 of secondary anti-rabbit antibody. Does anybody have experience with this CS antibody? Should I try others antibodies such the sigma M2?
Thank you very much!
Mercedes
I also think that anti-FLAG M1 monoclonal antibody (Purified IgG, Sigma-Aldrich) is useful.
Where is your FLAG tag and is there a single or triple repeat of the FLAG epitope? The location and number of repeats can dictate which antibody works well. In the past, for C or N terminal expression of a triple FLAG epitope (DYKDDDKx3) I have used Sigma's M2 antibody (mouse monoclonal affinity agarose for IP or M2 polyclonal from rabbit for western blots, 1:5000 dilution) and found it to be very successful.
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