> From: mamber at synapse.bms.com (Stephen Mamber)
> Subject: Re: CPRG
> Date: 12 Jun 1995 13:08:08 GMT
> Nntp-Posting-Host: 140.176.141.176
> X-Newsreader: WinVN 0.92.6+
>> In article <01HRFI55BQOY9X4U17 at CENTER.COLGATE.EDU>,
>BHOOPES at CENTER.COLGATE.EDU says:
>> > Can anyone steer me to a procedure for using this compound for yeast
> > beta-gals? I understand it is about 10 times more sensitive than
> > ONPG--anyone have any experience with this?
>> It's in the literature - check under the name David Eustice - he wrote
> a paper on it.
>> SWM
Below you will find something that is a bit more useful
(from Network Entrez),
best regards,
francis
--
| B.F. Francis Ouellette
||francis at ncbi.nlm.nih.gov
Biotechniques 11: 739-40, 742-3 (1991) [92232279]
A sensitive method for the detection of beta-galactosidase in transfected
mammalian cells.
D. C. Eustice, P. A. Feldman, A. M. Colberg-Poley,
R. M. Buckery & R. H. Neubauer
Viral Diseases Research, Du Pont Merck Pharmaceutical Co.,
Du Pont Experimental Station, Wilmington, DE 19880-0328.
A sensitive method has been developed for the detection of E. coli beta-
galactosidase in transfected HeLa cells. The chromogenic substrate, CPRG
(chlorophenol red-beta-D-galactopyranoside), was compared with ONPG (o-
nitrophenyl-beta-D-galactopyranoside) by kinetic analysis with purified beta-
galactosidase. The Km for CPRG was 1.35 mM and the Vmax was 21.4, whereas the
Km for ONPG was 2.42 and the Vmax was 41.1. CPRG at 8.0 mM (6-fold Km) gave 86%
of the Vmax and was used as the standard concentration for quantitation of
enzyme levels. The Vmax for CPRG was half that for ONPG, and chlorophenol red
has an extinction coefficient that is 21-fold higher than o-nitrophenol; these
factors make CPRG about 10-fold greater in sensitivity for the quantitation of
enzyme levels. The use of Nonidet P-40 to lyse the cells and the use of CPRG as
substrate permitted the rapid detection of low levels of enzyme production from
transfected human cells that could not be detected using ONPG.
MeSH Terms:
Chlorophenols
Cloning, Molecular
Galactosides
Genetic Techniques
Hela Cells
Human
Kinetics
Plasmids
Sensitivity and Specificity
Substrate Specificity
*Transfection
beta-Galactosidase/*analysis
Substances:
beta-Galactosidase (EC 3.2.1.23)
Plasmids
Chlorophenols
Galactosides
chlorophenol red galactopyranoside
