[Arabidopsis] About generation of specific antisera against plant protein

Thomas Leustek via arab-gen%40net.bio.net (by leustek from AESOP.Rutgers.edu)
Fri Jun 1 08:03:38 EST 2007

We have noticed this in many of the antisera we have produced. The 
only solution is to immunopurify the antibodies in the sera that you 
are interested in. There are many different protocols for immunopurification.

The reason for the non-specific reactions is uncertain. However, it 
should be noted that rabbits eat plants, therefore, the older they 
get, the more likely that they will have developed immunity against 
plant proteins. We have solved the problem of non-specific antibodies 
somewhat by using only very young rabbits and harvesting the bulk of 
the serum as soon as the titer is acceptable.

At 11:34 PM 5/31/2007, Chan Kam Ho wrote:
>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.
>Kam Ho
>Arab-gen mailing list
>Arab-gen from net.bio.net

Thomas Leustek
Rutgers University
Biotechnology Center for Agriculture and the Environment
Department of Plant Biology and Pathology
59 Dudley Road, Room 328
New Brunswick, New Jersey 08901-8520
Tel: 1(732)932-8165, ext 326
Fax: 1(732)932-0312
Mobile: 1(908)370-8696
LabWeb: http://aesop.rutgers.edu/~leusteklab/
BiotechWeb: www.biotech.rutgers.edu

also Director of the Rutgers Plant Biology Graduate Program
Web: www.plantbio.net

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