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!
-Kex2p-
Affinity purification is pretty good, you could try passing through a FPLC column to purify the antibody a bit first
-bob1-
QUOTE (Kex2p @ May 6 2008, 04:26 PM)
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!
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...
-The Bearer-