Purifying antibody

Allison Haggarty haggarty at med.mcgill.ca
Thu Aug 16 08:05:03 EST 2001



gs207 at yahoo.com wrote:
> 
> Hi
> 
> 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
precipitation 
on the whole serum. Then if you need to clean it up even more you can
run 
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.

Allison




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