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Immunoprecipitation protocol

Virginia Dress Dress at biosci.arizona.edu
Mon Jul 25 13:28:05 EST 1994

In article <rosto001.774909579 at maroon>, rosto001 at maroon.tc.umn.edu
(Alexander P Rostovtsev) wrote:
> In <bcguilf.25.0 at muccmail.missouri.edu> bcguilf at muccmail.missouri.edu (Guilfoyle Lab) writes:
> >Hi, 
> >I'm trying to study some protein-protein interactions using in vitro
> >translated HA-epitope-tagged proteins. I need a reliable protocol for 
> >doing immunoprecipitations from these in vitro translation reactions
> >using monoclonal 12CA5 which recognizes HA epitope. So far it looks like
> >I'm having high background problem coming from non-specific binding to
> >Protein A-Sepharose beads.
> 	Why do you use protein A-Sepharose binding monoclonal Abs rather
> poor? I usually use monoclonal Abs crosslinked to the protein G-Sepharose
> Alexander Rostovtsev

I believe the affinity of Protein A vs. Protein G for various antibodies 
depends on their _isotype_ NOT the fact that they are monoclonal (obviously
polyclonal antibody could have a mix of isotypes so this would not be a
Let me know if I am mistaken.  Check out the antibody book by Harlowe and
Cold Spring Harbor Press for hints on decreasing your background, I'm not
Protein G will solve your problem.  There should be something in there
buffers and preabsorbing the beads to minimize background.
Could the problem be in the translation reaction?  Could you be getting a
of premature products that are causing a high background?  What does a
blot look like vs. your immunoppt?



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