Where does mouse BRCA1 migrate on a western blot? - (Sep/28/2013 )
Does anyone have experience and success with detecting mouse BRCA1 by western blot? BRCA1 antibody datasheets indicate the protein should be around 220kd. I feel this specific to human because I think mouse is actually a smaller protein. I feel that mouse BRCA1 migrates around 200-210kd because I a using a new antibody with less background and the predominant band is around 205kd. Most antibodies produce a lot of non-specific bands so I am trying to figure out which band is correct. I have knocked down BRCA1 but with so many non-specific bands and not knowing where mouse BRCA1 migrates, it is hard to determine which band is correct. I also load at least 50ug protein fro whole cell lysate and have used nuclear lysates too.
The classic way to do this would be to get some peptide against the epitope of the antibody and then pre-incubate the antibody with the peptide. Finally use the pre-incubated antibody to probe a membrane, the abolished bands should be the specific ones.
However, I know that BRCA1 is particularly problematic with respect to background in humans, and I haven't tried it in mice, so can't really help there.