B-Galactosidase Assay Protocols

The assay for ß-galactosidase relies on the ability of the enzyme to catalyze the hydrolysis of ONPG (o-nitrophenyl-ß-D- galactopyranoside) to free o-nitrophenol, which absorbs light at 420 nm. In this protocol, extracts of cells transfected with a ß-galactosidase reporter plasmid are incubated with ONPG. - [Read Assay for ß-galactosidase in Extracts of Mammalian Cells]

This assay is used when working with phage vectors carrying the beta-gal gene. If the cloning event disrupts a normally functional copy of the gene in the vector the resulting plaques would appear clear in the assay. If the phages contain a functional beta-gal gene they will form blue rings around their plaques. Any strain which is not an overproducer of beta-gal will work as indicator host bacteria; a single chromosomal copy of the gene is not a problem. - [Read Assay for Phage Containing the Beta-galactosidase Gene]

Method describes how a crude extract is prepared, and the activity is normalized to the amount of protein assayed. This method is particularly suitable for comparing cells that are grown under very different conditions or that have different genetic backgrounds. - [Read Assay of ß-Galactosidase in Yeast: Assay of Crude Extracts]

Method described indicates how cells are lysed by a simple freeze/thaw protocol and treated with a chemiluminescent substrate. - [Read Assay of ß-Galactosidase in Yeast: Freeze/Thaw Assay by Chemiluminescence in 96-Well Plates]

In the method described here, the cells are lysed by a simple freeze/thaw protocol and . . . - [Read Assay of ß-Galactosidase in Yeast: Freeze/Thaw Assay by Chemiluminescence in Microcentrifuge Tubes]

In the method described here, the cells are permeabilized to allow the substrate to enter the cells and the activity is normalized to the number of cells assayed. - [Read Assay of ß-Galactosidase in Yeast: Permeabilized Cell Assay]

There are two basic methods for the in vitro assay of B-galactosidase activity from yeast. They differ mainly in the method of preparing the material for assay. Both methods are described with accompanying protocols. Method I: Assay of Crude Extracts includes: Yeast Cell Growth; Yeast Cell Harvest; B-gal assays; Bradford Assays. Method II: Permeabilized cell assay. - [Read Assay of β-Galactosidase in Yeast Protocol]

The recommended amount of RSV-ß-Galactosidase plasmid to use for transfection of cells (60 mm or 100 mm dish) is 1-2 µg. The optimal amount of plasmid DNA will be determined by the efficiency of transfection , which is very dependent upon the particular cell line and transfection protocol. - [Read B- Galactosidase Assay Protocol]

Protocol for B-Gal filter assay. Includes Z buffer and X-Gal solution. - [Read B-GAL Filter Assay Protocol]

Basic protocol for a Beta-gal assay. - [Read Beta-gal Assay Protocol]

Protocol for Beta-galactosidase reporter gene assay (liquid form). - [Read Beta-galactosidase Reporter Gene Assay (Liquid Form)]