Griffith Kevin L, Wolf Richard E
Department of Biological Sciences, University of Maryland Baltimore County, 1000 Hilltop Circle, Baltimore, MD 21250, USA.
Biochem Biophys Res Commun. 2002 Jan 11;290(1):397-402. doi: 10.1006/bbrc.2001.6152.
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.
我们描述了一种用于测量细菌中β-半乳糖苷酶活性的高通量方法。该方法独特之处在于，包括细菌生长和细胞通透化在内的所有操作均以96孔板形式进行。通过氯仿/十二烷基硫酸钠处理直接在96孔板中使细胞通透化，然后转移至96孔微孔板中，按照米勒[J. H. 米勒（1972年）《分子遗传学实验》，冷泉港实验室出版社，纽约冷泉港]所述方法进行β-半乳糖苷酶活性的标准比色测定。用酶标仪收集吸光度数据，并使用Microsoft Excel电子表格进行分析。通过高通量方法获得的β-半乳糖苷酶比活性值与通过传统单管米勒法获得的值相同。因此，用该方法获得的值可以表示为米勒单位，并可直接与文献中报道的米勒单位进行比较。用于酶特异性活性通透化和测定的96孔板形式，连同12通道和重复移液器的使用，能够在8小时内高效处理数百个样品。
