??CAT assays??

Uttam Rajbhandary bhandary at wccf.mit.edu
Fri Apr 22 19:45:00 EST 1994


In article <ander-110494141000 at romulus.mayo.edu>, ander at rcf.mayo.edu (Robert A. Anders) writes...
>Does anyone use 4 mM Acetyl-CoA in their CAT assays?  I am currently using
>0.4 mM [final concentration] and experiencing some trouble.  I was
>wondering if the higher concentration is routinely used.
> 
>Thanks for any replys   RAA

What kind of "trouble" are you having?  What kind of extracts?

We typically use about 0.6 mM final, using a 50 ul rxn, and up to 30-60 ug of 
COS-1 or CV-1 cell extract protein.  Rxn times are typically for 30 minutes.  
When incubating longer, a second aliquot of Acetyl-CoA is added at the 
begining of each 30 minute period.  This is because the acetyl-coA is broken 
down by enzymes in the cell extracts.  An alternative is to head an aliquote 
of the extract at 65C for 15 minutes and spining out the insoluble material 
(you'll need to remeasure the protein content).  This method has been 
recommended in protocols that use 3H-acetyl-CoA rather than the 14C-CAM.  We 
have found that we get increased activity using the 14C-CAM using heat treated 
extract.  I say use an aliquote because if you are going to measure another 
protein (say, b-gal), that protein may not be stable to the heat treatment.

It is claimed that heating the extract allows for VERY long incubation times, 
still giving a linear response (required if promoter activities aren't high; 
using COS-1 and SV40-based CAT expression systems makes one spoiled ).

If you haven't already seen these references, I recommend them:

Newmann, Morency, Russian  1987  Biotechniques 5 (5), 444-447
Sleigh 1986  Analytical Biochem. 156, 251-256 (original)
Hollon and Yoshimura (1989) Anal. Biochem. 182, 411-418 (use up to 100ug of 
protein!).

You might be interested in this too: 

Alter and Subramanian 1988 Biotechniques 6 (6) 526-530 (direct measurement of 
cat activity by incubation of cat-expressing cells in medium containig 
chloramphenicol).  It DOES work.


Regards

Harold Drabkin  c/o bhandary at wccf.mit.edu




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