haggarty at med.mcgill.ca
Thu Aug 16 08:05:03 EST 2001
gs207 at yahoo.com wrote:
> I am new to the field of Immunology etc, so please excuse me if I ask
> something stupid. I want to use an antibody in immunoprecipitation
> assays . All I have been provided with are bleeds from rabbit immunized
> with peptide from the protein of interest. The peptide has a cysteine
> at amino terminus. I have been told that I need to purify that antibody
> with that peptide before using it. I have no idea how to go about it.
> I would really appreciate any help with this. If you can guide me to a
> published protocol (better if you can provide me with one you have used)
> and/or any kits I can use. Thanks a lot.
The easiest way to start would be to do an ammonium sulphate
on the whole serum. Then if you need to clean it up even more you can
it over a Protein A column to retain only the immunoglobulins. Both are
standard procedures that you can find in an immunological methods book.
For a good negative control in your immppts you can do the same
procedures in parallel on normal rabbit serum - ideally preimmune serum
from the rabbit that you have the bleeds from.
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