Measuring β-Galactosidase Activity in Bacteria: Cell Growth, Permeabilization, and Enzyme Assays in 96-Well Arrays
| Autor: | Kevin L. Griffith, Richard E. Wolf |
|---|---|
| Rok vydání: | 2002 |
| Předmět: |
Time Factors
Biophysics Bacterial growth Biology Polypropylenes behavioral disciplines and activities Biochemistry Bacterial cell structure Escherichia coli Molecular Biology Chromatography Bacteria Sodium Dodecyl Sulfate Galactosidase activity Cell Biology beta-Galactosidase biology.organism_classification Molecular biology Enzyme assay Microplate Reader Spectrophotometry biology.protein Specific activity Chloroform Cell Division Software Plate reader |
| Zdroj: | Biochemical and Biophysical Research Communications. 290:397-402 |
| ISSN: | 0006-291X |
| DOI: | 10.1006/bbrc.2001.6152 |
| Popis: | We describe a high-throughput procedure for measuring β-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 β-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 β-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. |
| Databáze: | OpenAIRE |
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