Need help with CAT assays in bacteria
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
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.
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).
More information about the Methods