Need help with CAT assays in bacteria
Sarah K. Highlander, Ph.D.
sarahh at bcm.tmc.edu
Thu Aug 15 16:33:45 EST 1996
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
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
One Baylor Plaza || ||
Houston, TX 77030-3498 || ||
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