Did you try a Western with your CRUDE SERUM and the peptide column-purified
antibodies (separately), against the original peptide, the BSA-coupled
peptide and the corresponding proteins? (I know that it sounds trivial, but
just in case...). Also, if your proteins are not highly expressed in the
cells, it would help to have some transfected cells checked (if available).
One possibility is that you lost the antibodies on the peptide column. To
attach the peptide to the column, you have probably masked some epitopes
(and the antibodies against those epitopes went down with the flow-through).
In addition, the best anti-peptide IgG molecules within your polyclonal
serum will attach so strongly to the peptide on the column that it may be
difficult to elute them afterwards. I would suggest to check first the
crude serum (if you have any left), and if it does not work, then go back to
earlier steps.
Assuming the crude sera were OK against the peptides (otherwise it would not
have made sense to sacrifice the animals), your 20% final detection rate is
low, especially for a Western (the proteins are denatured during the
SDS-PAGE, therefore during renaturing on the membrane after the transfer,
you should have had all kinds of folding going on, and the part
corresponding to your peptide should have been exposed to the antibodies,
even if it were hidden in the native protein).
Regards,
wagner
-----Original Message-----
From: Association of Biomolecular Resource Facilities
[mailto:abrf-request@aecom.yu.edu]On Behalf Of Dick Wicks
Sent: 3 aošt, 1999 07:47
To: Recipients of ABRF List
Subject: Antipeptide antibodies
Dear ABRFers:
After seeing the post on the anti-peptide antibodies, I was
wondering if anyone could give me feedback on our experience with
making these. We made 12 mer peptides from 85 different proteins
(selected for hydrophilicity, antigenicity, etc using DNASTAR
program). Coupled to BSA via the terminal cysteine that we added
using SMCC, then injected into rabbits. The resulting antibodies
were purified on peptide columns. Only about 20% of the
antibodies reacted with the respective proteins in Western blots
under reducing conditions. Any idea whether this is unusual?
Thanks very much.
Dick Wicks