Need help with CAT assays in bacteria

Mikhail Alexeyev malexeyev at biost1.thi.tmc.edu
Thu Aug 15 17:13:27 EST 1996


In article <321397B9.69D7 at bcm.tmc.edu>, sarahh at bcm.tmc.edu wrote:

> 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
> -- 
 
 1. Quantitative nonradioactive CAT assays using fluorescent BODIPY
      1-deoxychloramphenicol substrates.   Lefevre CK.  Singer VL.  Kang HC.
      Biotechniques.  19(3):488-93, 1995 Sep.

   2. Increasing the sensitivity of a common CAT assay.   Cassinotti P.
      Weitz M.
      Biotechniques.  17(1):36, 38, 40, 1994 Jul.

   3. An improved fluor diffusion assay for chloramphenicol acetyltransferase
      gene expression.   Purschke WG.  Muller PK.
      Biotechniques.  16(2):264-5, 268-9, 1994 Feb.

A friend of mine spoke highly of method of Neuman et. al. BioTechniques 5
(1987) 444-447 (she used it on E. coli).

Good luck,
M.A.



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