jakami at ucdavis.edu
Tue Aug 21 14:33:54 EST 2001
The best method I know of for purifying the IgG fraction (antibodies) is
to precipitate it with polyethylene glycol. First centrifuge at 4C to
remove clots and cold insoluble globulins then, to the supernatant, add 50%
Polyethylene Glycol (MW 8000) , dropwise, while stirring, to a final conc.
of 13%. Spin out the precipitate (the IgG fraction) discard the supernatant
and dissolve the pellet to the original volume with PBS. Repeat the
precipitation step. The resulting fraction is >95% pure with an almost
quantitative yield and full activity. (Ammonium Sulfate, in my hands, gives
a ~50% pure product with a loss of both protein and activity). Any residual
PEG does not seem to have any effect on subsequent reactions. Note: The
purified fraction, now devoid of the other protective proteins in serum
(proteases, anti-trypsin, complement etc) should be stored cold, with .02%
sodium azide as a preservative.
This material can then be used for the affinity chromatography method you've
been advised to use. The best book I ever seen on immunochemistry was
called, simply "Antibodies : a laboratory manual /, edited by Ed Harlow,
David Lane. Cold Spring Harbor, NY : Cold Spring Harbor Laboratory, 1988.".
It is a cookbook-like methods manual along the same lines as Maniatis.
Hope this helps, good luck.
Dept. of Agronomy and Range Science
University of California
Davis, California 95616
email: jakami at ucdavis.edu
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.
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