immunoprecipitation

Ian A. York iayork at panix.com
Tue Sep 14 17:49:51 EST 1999


In article <37DEBAFB.2DB43E07 at med.unc.edu>,
Caroline Szymeczek-Seay  <css at med.unc.edu> wrote:
>
>label the cells, immunoppt with anti-protein X, and run the reaction on
>a gel, flurograph, and expose.  This should give me, if protein X
>interacts with X, Y, and Z, three different bands with intensities equal
>to their affinity for X.  It won't tell me what proteins those bands

If I follow you, you can't use this system to measure affinity.  The
intensities of the ands will be related to the proteins' affinities, and
also (and probably much more importantly) to their concentrations.  To
measure affinity you need to know the concentration--even if protein Z had
a much lower affinity for X than Y, if it is present at 100 times the
concentration of Y it may still look like the dominant binding partner.

As you described the experiments, you have no idea of the relative
concentrations of each protein, and determining this is not a trivial
task.  (Keep in mind, too, questions of subcompartmentalization and
concentrations in a microenvironment.)

If you really need to know affinities, you probably need to do the
experiment in vitro in a defined system.

Ian 

-- 
    Ian York   (iayork at panix.com)  <http://www.panix.com/~iayork/>
    "-but as he was a York, I am rather inclined to suppose him a
     very respectable Man." -Jane Austen, The History of England



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