Western Blot Background - Having high backgrounds on western blotting due to antibody issues. (May/06/2008 )
I am having high background because non-specific antibodies were also made in the rabbit in addition to the specific antibody for s.s. Kex2p (Protein of interest). I heard about fixing this through subtraction hybridization then affinity purification, but it seems like an older method. Does anyone know a more modern procedure or is the hybridization the best route? Thanks to all!
Affinity purification is pretty good, you could try passing through a FPLC column to purify the antibody a bit first
most simple is a two- to four-time re-usage of the same antibody solution for a Western; the ratio of specific to unspecific Ab´s increases...