Strategy for Antibody production

Edmundo Castro ecastro at PHYSCI.UCLA.EDU
Thu Aug 22 15:57:34 EST 1996


We are trying to produce antibodies against a murine Gq coupled cell
surface receptor to use for immunocytochemical and histochemical
staining.  Since there are several other members in this family we were
advised that in order to minimize the potential for crossreactivity we
should not use the full-length protein.
We decided to use the carboxy end to immunize the rabbit since there was
more aa divergence here than in other regions.  The 40 C-terminal most
aa were expressed in bacteria via an Invitrogen pRSET vector.  Antigen
was purified by SDS-PAGE after islolation by Ni column.  By Western, our
antisera recognizes purified antigen and recognizes a protein of
appropriate size (50 kD) in appropriate tissue up to a 1:5000 or
1:10,000 dilution.  Unfortunately, our antibody reacts even more
strongly to a higher molecular weight protein (60 kD) that is expressed
in most cell types.  Absorbing the Ab by using acetone powders (1% or
10%) has been ineffective.  Affinity purification by using a blotted
strip of purified antigen also did not remove the larger band.

We are getting ready to try to immunize again and I wonder whether a
different strategy would be more effective:  (1) Should we try the
full-length protein?  (2) Should we immunize with the carboxy peptide in
the first 2 shots and then use native protein as  a booster?  (3) Try
more rabbits since we have only injected 1 so far.  (4) Express a
different region of the protein in bacteria as new antigen.

Thanks to all in advance!

EC



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