Need help with CAT assays in bacteria

[Sarah K. Highlander, Ph.D.]

I am trying to assay cat activity in bacterial extracts. This is my 
first go-round and could use some help. I am seeing activity using a 
plasmid-borne cat gene but the background for non-plasmid containing 
strains seems to be very high. I am concerned that the DMP-acetyl CoA 
premix is very yellow to begin with and is obscuring the development of 
color as the reaction proceeds. Is this mixture supposed to be bright 
yellow? I believe that I made it correctly following the method of Shaw 
(100 mM Tris, pH 7.8, 0.1 mM acetyl-CoA from yeast, 0.4 mg/ml DTNB).
My reaction volume is 5 ml of this mixture to which I add 0.1 ml of a 
clarified sonicated extract from a 10x concentrated overnight culture. I 
am allowing the reaction to proceed for about an hour at 37°C.

Can anyone offer any help? I spent some time in library and could not 
find much more information beyond Shaw's Meth Enzymol 43:737-755. 
Additional references to specific procedures would also be helpful

Thanks,

Sarah
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_______________________________________________________________________
Sarah K. Highlander, Ph.D       
Department of Microbiology   (__==__)        email: sarahh at bcm.tmc.edu
  & Immunology                 (oo)       *  Voice: 713-798-6311
Baylor College of Medicine      \/-------/   FAX    713-798-7375
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