Schaefer Jorrit, Jovanovic Goran, Kotta-Loizou Ioly, Buck Martin
Department of Life Sciences, Faculty of Natural Sciences, Imperial College London, London SW7 2AZ, UK.
Department of Life Sciences, Faculty of Natural Sciences, Imperial College London, London SW7 2AZ, UK.
Anal Biochem. 2016 Jun 15;503:56-7. doi: 10.1016/j.ab.2016.03.017. Epub 2016 Mar 29.
Historically, the lacZ gene is one of the most universally used reporters of gene expression in molecular biology. Its activity can be quantified using an artificial substrate, o-nitrophenyl-ß-d-galactopyranoside (ONPG). However, the traditional method for measuring LacZ activity (first described by J. H. Miller in 1972) can be challenging for a large number of samples, is prone to variability, and involves hazardous compounds for lysis (e.g., chloroform, toluene). Here we describe a single-step assay using a 96-well microplate reader with a proven alternative cell permeabilization method. This modified protocol reduces handling time by 90%.
从历史上看，lacZ基因是分子生物学中最广泛使用的基因表达报告基因之一。其活性可以使用人工底物邻硝基苯基-β-D-吡喃半乳糖苷（ONPG）进行定量。然而，测量LacZ活性的传统方法（由J. H. 米勒于1972年首次描述）对于大量样本可能具有挑战性，容易出现变异性，并且涉及用于裂解的危险化合物（例如氯仿、甲苯）。在这里，我们描述了一种使用96孔微孔板读数器的单步测定法以及一种经过验证的替代细胞通透化方法。这种改进的方案将处理时间减少了90%。
