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A sensitive method for the detection of beta-galactosidase in transfected mammalian cells. 1991

D C Eustice, and P A Feldman, and A M Colberg-Poley, and R M Buckery, and 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.

UI MeSH Term Description Entries
D007700 Kinetics The rate dynamics in chemical or physical systems.
D010957 Plasmids Extrachromosomal, usually CIRCULAR DNA molecules that are self-replicating and transferable from one organism to another. They are found in a variety of bacterial, archaeal, fungal, algal, and plant species. They are used in GENETIC ENGINEERING as CLONING VECTORS. Episomes,Episome,Plasmid
D002733 Chlorophenols Phenols substituted with one or more chlorine atoms in any position. Chlorophenol,Hydroxychlorobenzenes
D003001 Cloning, Molecular The insertion of recombinant DNA molecules from prokaryotic and/or eukaryotic sources into a replicating vehicle, such as a plasmid or virus vector, and the introduction of the resultant hybrid molecules into recipient cells without altering the viability of those cells. Molecular Cloning
D005697 Galactosides Glycosides formed by the reaction of the hydroxyl group on the anomeric carbon atom of galactose with an alcohol to form an acetal. They include both alpha- and beta-galactosides.
D005821 Genetic Techniques Chromosomal, biochemical, intracellular, and other methods used in the study of genetics. Genetic Technic,Genetic Technics,Genetic Technique,Technic, Genetic,Technics, Genetic,Technique, Genetic,Techniques, Genetic
D006367 HeLa Cells The first continuously cultured human malignant CELL LINE, derived from the cervical carcinoma of Henrietta Lacks. These cells are used for, among other things, VIRUS CULTIVATION and PRECLINICAL DRUG EVALUATION assays. Cell, HeLa,Cells, HeLa,HeLa Cell
D006801 Humans Members of the species Homo sapiens. Homo sapiens,Man (Taxonomy),Human,Man, Modern,Modern Man
D001616 beta-Galactosidase A group of enzymes that catalyzes the hydrolysis of terminal, non-reducing beta-D-galactose residues in beta-galactosides. Deficiency of beta-Galactosidase A1 may cause GANGLIOSIDOSIS, GM1. Lactases,Dairyaid,Lactaid,Lactogest,Lactrase,beta-D-Galactosidase,beta-Galactosidase A1,beta-Galactosidase A2,beta-Galactosidase A3,beta-Galactosidases,lac Z Protein,Protein, lac Z,beta D Galactosidase,beta Galactosidase,beta Galactosidase A1,beta Galactosidase A2,beta Galactosidase A3,beta Galactosidases
D012680 Sensitivity and Specificity Binary classification measures to assess test results. Sensitivity or recall rate is the proportion of true positives. Specificity is the probability of correctly determining the absence of a condition. (From Last, Dictionary of Epidemiology, 2d ed) Specificity,Sensitivity,Specificity and Sensitivity

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