Immunoprecipitation using proteinA agarose

Eddie Diehl diehl001 at mc.duke.edu
Sun Feb 4 12:49:03 EST 1996


In article <3111B4FC.6C73 at vision.postech.ac.kr>, Lab, of,
SignalTransduction, Dep.of, Life, Science, POSTECH, San31, HyoJaDong,
PoHang, South, Korea wrote:

> Hi! everyone.
> I am graduate student in south korea.
> I want to know tip that can reduce non-specific association of cell 
> extract to protein A agarose in immunoprecipitation experiment.
> I routinly add antibody solution to pre-cleared mammalian cell 
> extract and then add protein A agarose.
> In my opinion, nonspecific binding is due to washing buffer.
> My washing buffer contain 1% Triton X-100 and 180mM NaCl.
> Please, give me solution.
> Thanks in advance.
> My email address is
> pgs at vision.postech.ac.kr
> Myung Jong Kim


It would help if I knew more about the protein(s) that you are trying to
immunoprecipitate. Are you trying to co-precipitate protein complexes? If
not, one can use more stringent conditions as a rule.

I used to wash in 1% (w/v) sodium deoxycholate and .1% (v/v) Triton X100.
You can also pre-bind your antibody to the protein A, but as long as
everything is done in the cold this shouldn't reduce non-specific protein
binding. Some people can get away with adding the detergents and maybe
some salt to their homogenates before adding the antibody, which should
cut down on non-specific binding. You can also try pre-incubating your
homogenates with protein A, then adding your antibody and completing the
immunoprecipitation.

Best of luck!

Eddie Diehl
diehl001 at mc.duke.edu



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