We are discussing, in our lab, our plans for raising addition anti-peptide
antibodies against regions of the thromboxane A2 reeceptor. We've already
produced two (one against a sequence on the 1st extracellular loop, and
one on the intracellular C-terminal) that we use for Western blots, &c. We
would like to produce some Fab fragments as a means to explore the areas of
the receptor that may or may not couple to various G proteins. Since these
regions have not been identified, we were pouring over some antigenicity
analyses and hydrophobicity plots to see which 10-12 residue sequences would
be best. We came up with three, and then my advisor asked a rather interesting
question: "Can we immunize a single rabbit with a mixture of all three
(KLH-conjugated) peptides?"
He (and I) see the relative advantages in this method as being the following:
1.) Use fewer rabbits (and therefore, fewer immunizations, etc.)
2.) This "shotgun" approach will (if it works) allow us to screen three
antibodies at once by treating saponin-permeabilized platelets with
Fab fragments and looking for changes in GTP-binding (or whatever
endpoint) in response to TXA2 analogues.
3.) We can separate the antibodies using affinity chromatography if
necessary.
We do question, however, whether or not a rabbit will produce all three
antibodies, and whether or not there will be a problem since the antigens
will be relatively similary (KLH +/- a particular 10-12 residue sequence).
So I turn to the 'net for feedback. Any ideas on this approach? Has
anyone tried it? Any suggestions? I'll post a summary to the group(s)
if requested.
Thanks for your advice!
-- Sam
--
Samuel C. Blackman ! InterNet : scb1 at midway.uchicago.edu
MD/PhD 2/7 (Pharmacology) ! Disclaimer : Who cares what I say, I'm a student!
5513 S. Cornell Ave. #1 ! Quote : I'm not a doctor, but I play one on TV.
Chicago, IL 60637-1914 ! Phone : 312/752-1082 (h) Fax: 312/996-1225
