peptide binding to plates&/or membrane
dspinella at chugaibio.com
Thu Jan 15 22:49:57 EST 1998
> Hi everyone.
> I need to bind a peptide to a plate or nirtocellulose membrane for use
> in ELISA or western. The problem is, however, I would like to do so
> without covalent coupling to a carrier (I have done this already to
> check the antiserum).
> Any suggestions???
> Alison Leakey
> North Queensland Cinical School Research Laboratory
Have you tried simple direct adsorption of your peptide to a plastic
plate (styrene or PVC) or to nitrocellulose? This is pretty hit or miss
depending on the peptide in question, but its worth a try as some
peptides DO adsorb to plastic just fine and behave in standard ELISAs
just like typical protein antigens. In general, the more hydrophobic
the peptide, the better your chances of getting this to work. Sometimes
drying the peptide solution down in the wells using a speed vac helps as
does varying the pH of the coating buffer. Failing this, there are
commercially available microtiter plates that have NHS ester groups that
will react with free amines to covalently capture your peptide (try Nunc
or Pierce Chemical). Another thing that can be tried is to ask your
peptide synthesis service to provide you the peptide uncleaved from the
resin -- in which case you already have a solid phase which can simply
be spun down inside microtiter wells during the washing steps.
Assuming that all this fails, you can always conjugate the peptide to a
different protein carrier than was used for immunization (I guess I'm
assuming that you did conjugate the peptide to a carrier for
immunization). As long as the anti-carrier antibodies in the serum
don't cross-react with the new (coating) carrier -- which is easily
verified by running controls of unconjugated carrier, any response in
the ELISA must be due to anti-peptide antibodies. Or did I totally
misinterpret your question and what you're trying to do??
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