CAT assay - best method?

[Stephen R. Lasky]

In article <4946te$87q at saba.info.ucla.edu>, bbraun at ucla.edu wrote:

> I'm sure this is a common question, so FAQ pointers are welcome.
> 
> What is the best way do do CAT assays?
> Concerns include:
> 
> 1. sensitivity
> 2. reproducibility
> 3. convenience - e.g. 50 samples or more in one sitting
> 4. compatibility of lysate with B-gal or Luciferase assays
> 5. anything else?
> 
> Note that cost, although important, is not a priority.  I am
> currently leaning towards an ELISA kit like the one
> Boehrringer sells.
> 
> Any suggestions?
> 
> Benjamin Braun

I've been using the B-M Cat-elisa kit and like it better than TLC-cat
assays.  It is very sensitive and reproducible but labor intensive.  I
think that the detergent lysis protocol is not compatible with b-gal
assays, but I have found that although the kit instructions are slightly
different, the CAT-elisa  protocol will work for B-M's GH-elisa assays, so
if you can cut down to 48 samples at a time you can do both assays at the
same time in the same plate rack (96 samples) and then read them at the
same time in your plate reader.   I found, however, that  using  0.25
micrograms of  CMV-GH vector for the internal control I have to dilute the
superantants by 10 fold to get into a reasonable linear range at the same
time as the CAT assay.  If you used the TK-GH vector, you might not have
to do this.

SRLasky

-- 
Stephen R. Lasky Ph.D.
Section of Experimental Therapeutics
Roger Williams Medical Center
Providence, RI 02908
Phone: 401-456-5672      Fax: 401-456-6569
e:mail: Stephen_Lasky at brown.edu