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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
(deleted)
> 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
a
polyclonal antibody could have a mix of isotypes so this would not be a
problem)
Let me know if I am mistaken.  Check out the antibody book by Harlowe and
Lane
Cold Spring Harbor Press for hints on decreasing your background, I'm not
sure
Protein G will solve your problem.  There should be something in there
about
buffers and preabsorbing the beads to minimize background.
Could the problem be in the translation reaction?  Could you be getting a
lot
of premature products that are causing a high background?  What does a
Western
blot look like vs. your immunoppt?

Ginnie

 



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