CAT assays (in cell culture media)

J. Kaufmann kaufmann at cmu.unige.ch
Mon Nov 14 10:09:19 EST 1994


I have made a fusion protein with human proinsulin and CAT and I'm
expressing it in AtT20 cells. I want to determine if this protein is
targetted to the regulated secretory pathway or to the constitutive pathway
(or if it is degraded in the cell). I stimulate cell secretion with IBMX
and forskolin during an hour to determine if I can stimulate secretion of
my fusion protein.

I can detect nice levels of expression of CAT in cell extracts but I can't
detect anything in cell media. I have observed that CAT activity is greatly
inhibited in DMEM or in KRB. It is less inhibited in another release medium
containing NaCl, KCl and Hepes, however the addition of calcium seems to
inhibit CAT activity (and cells don't secrete anything in absence of
calcium). It is also not possible to add EGTA for the CAT assay (CAT
activity is almost abolished in presence of EGTA).

I perform my CAT assays by diffusion of acetylated products in the
scintillation cocktail during the CAT assay, and another problem is the
volumes of media that I have to use. Extraction is much less efficient in
1ml of medium compared to 30µl of cell extract.

Has anybody tried to perform CAT assays in cell media? 

I need your advice!

Thank you!


-- 
Jocelyne Kaufmann
Laboratoires de Recherche Louis Jeantet
Centre Medical Universitaire
1, rue Michel Servet
CH-1211 Geneve 4
Switzerland
e-mail: kaufmann at cmu.unige.ch



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