Best epitope for IF with a poor-expresser - (Jan/13/2015 )
I am attempting to use IF on a poorly expressing protein in Helas and have been having trouble with high levels of background using Flag and the M2 antibody from sigma. Based on the localization of the background, it looks like Ppm1b that I have read cross-reacts with M2. As my protein of interest is also cytoplasmic, this is an issue.
Is there an epitope/antibody combination that is generally considered "best" for looking at low-abundance proteins?
Flag is usually pretty good and highly specific. I would avoid His and GST tags for IF, as the antibodies tend to be a bit messy, but most of the others seem to work well. You could also try a non-antibody system such as streptavidin/biotin.
I know this is not what you asked, but if your study allows it, you can also think of expressing the protein in different cells or same cells but different promoter to increase the expression levels.