We have been trying to produce specific polyclonal antisera against our
Arabidopsis proteins by injecting the rabbit using the recombinant proteins
(full-length or part of the sequence) expressed from E.coli.
however, we notice that using our western system, we found that the
post-immune sera usually recognize many many bands which are absent when we
use pre-immune sera to probe the same protein (wild-type extract like leaf.,
root, stem). We tried to use ECL and AP system and also amplify our signal/
We tried to block the membrane using 10% non-fat milk overnight and also
tried to purify the Antibodies by protein A chromatography. but multiple
bands appear. If we decrease the amount of protein or use very diluted
antisera (1:10000), we can't see any band,...
Are there anyone with similar experience to share? Thanks in advance.