β-Galactosidase Assay Services
Cell-based reporter gene testing provides another popular and cost-effective HTS platform for GPCR screening. β-galactosidase is one of the commonly used reporters that are enzyme proteins with activities linked to a variety of colorimetric, fluorescent, or luminescent readouts. Creative Biolabs has expertise and capabilities in providing a high-quality β-Galactosidase assay for your drug discovery.
Overview of β-Galactosidase Assay
The Escherichia coli lacZ gene (encoding β-galactosidase) is a reporter gene widely used in bacteria. Scientists routinely measure lacZ expression in bacterial cell-based two-hybrid assays to determine the relative strengths of various protein-DNA and protein-protein interactions. It is predominantly used as an internal control during transient transfection experiments. The activity of β-galactosidase is widely used as a marker phenotype in several biological systems. The availability of substrates other than lactose whose cleavage releases compounds that can be easily detected allows assay of enzyme activity on both a qualitative and quantitative basis.
Protocol of the β-Galactosidase Assay
- Cell Lysis
1) Remove growth medium from transfected cells.
2) Pellet the cells in a microcentrifuge at low speed (8000 rpm) for 1 min. Remove supernatant and wash with 1 mL of PBS. Pellet the cells again and remove the supernatant.
3) Suspend the cell pellet in two volumes of 0.25 M Tris-Cl, pH 8.0. Vortex.
4) Freeze the solution in a dry ice/ethanol bath. Thaw in a 37°C water bath. Repeat the freeze-thaw cycle three times to ensure complete cell lysis.
5) Chill the samples on ice. Pellet the cell debris in a microcentrifuge at 14,000 rpm for 5 min. Transfer the supernatant to a new tube.
6) Perform a Bradford Assay to determine the overall protein concentration of each lysate. Use 25 μg of each lysate for the β-galactosidase assay.
- β-Galactosidase Assay
1) Set up reaction tubes for each cell lysate.
2) Incubate at 37°C until a faint yellow color becomes apparent, at least in the tubes expected to contain the most activity.
3) Read the absorbance at 420 nm.
Fig.1 Overview of 96-well format β-galactosidase assay. (Thibodeau, 2004)
Advantages of β-Galactosidase Assay
- β-gal was found to be marginally more effective in providing a signal in the context of weak enhancers and to be particularly helpful in high-resolution histochemical analysis. In conjunction with more sensitive.
- β-gal activity is readily assessed in vitro or in vivo, in hosts as evolutionarily diverse as bacteria, yeast, and mammals, its induction has become a standard means of assaying clonal insertion, transcriptional activation, protein expression, and protein interaction.
Creative Biolabs is an outstanding, independent USA company offering quality products and first-class customer service to leading pharma, biotech, contract research organizations, and universities worldwide. We can meet requirements in different stages with rapid turn-around time, and 100% on-time delivery. If you are interested in our β-galactosidase assay services, please feel free to contact us for more.
Reference
- Thibodeau, S.A.; et al. High-throughput β-galactosidase assay for bacterial cell-based reporter systems. Biotechniques. 2004, 36(3): 410-415.
