|Measuring beta-galactosidase activity in bacteria: cell growth, permeabilization, and enzyme assays in 96-well arrays.
|Year of Publication
|Griffith KL, Wolf RE
|Biochem Biophys Res Commun
|2002 Jan 11
|Bacteria, beta-Galactosidase, Biochemistry, Cell Division, Chloroform, Escherichia coli, Polypropylenes, Sodium Dodecyl Sulfate, Software, Spectrophotometry, Time Factors
We describe a high-throughput procedure for measuring beta-galactosidase activity in bacteria. This procedure is unique because all manipulations, including bacterial growth and cell permeabilization, are performed in a 96-well format. Cells are permeabilized by chloroform/SDS treatment directly in the 96-well blocks and then transferred to 96-well microplates for standard colorimetric assay of beta-galactosidase activity as described by Miller [J. H. Miller (1972) Experiments in Molecular Genetics, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY]. Absorbance data are collected with a microplate reader and analyzed using a Microsoft Excel spreadsheet. The beta-galactosidase specific activity values obtained with the high-throughput procedure are identical to those obtained by the traditional single-tube method of Miller. Thus, values obtained with this procedure may be expressed as Miller units and compared directly to Miller units reported in the literature. The 96-well format for permeabilization and assay of enzyme specific activity together with the use of 12-channel and repeater pipettors enables efficient processing of hundreds of samples in an 8-h day.
|Biochem. Biophys. Res. Commun.