[Arabidopsis] About generation of specific antisera against plant protein

Chan Kam Ho via arab-gen%40net.bio.net (by h0204121 from gmail.com)
Thu May 31 22:34:48 EST 2007


Dear all,

 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.

Thanks
Kam Ho


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