Measuring beta-galactosidase activity in bacteria: cell growth, permeabilization, and enzyme assays in 96-well arrays.
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.
Griffith
Kevin L
KL
Department of Biological Sciences, University of Maryland Baltimore County, 1000 Hilltop Circle, Baltimore, MD 21250, USA.
Wolf
Richard E
RE
D001419:Bacteria / Q000201:enzymology*; D001671:Biochemistry / Q000379:methods*; D002455:Cell Division; D002725:Chloroform / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology; D011126:Polypropylenes / Q000737:chemistry; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology; D012984:Software; D013053:Spectrophotometry; D013997:Time Factors; D001616:beta-Galactosidase / Q000032:analysis* / Q000378:metabolism*
D011126:Polypropylenes; D012967:Sodium Dodecyl Sulfate; D002725:Chloroform; D001616:beta-Galactosidase