Removal of factor X from reaction

[Tom Morrison]

In article <2ovmk2$3uk at network.ucsd.edu>, T. S. Pillay <tpillay at ucsd.edu>
wrote:

> Does anyone know of an easy way to remove Factor x from a cleavage
> reaction after a fusion protein has been cleaved.

I am also worried about Xa staying with my cleaved protein because Xa
cleaves the kinase I plan to test my purified protein with.  

My first try is to use conditions where the Xa loses any significant
activity by the end of the digest.  (In my hands, Promega's and
Boehringer's Xa seem to lose their activities after 5 hrs and 2 hrs
respectively.)

If this fails, I purchased some Antithrombin III, a high affinity Xa
inhibitor, from Boehringer.  This should kill any remaining Xa activity,
and hopefully not inhibit my reactions.

I'm in the process of testing the above ideas.

-- 
Tom Morrison                                      Department of Biology
morrison at bioscience.utah.edu                      University of Utah