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A Saccharomyces cerevisiae cell‐based quantitative β‐galactosidase assay compatible with robotic handling and high‐throughput screening
Author(s) -
de Almeida Rogerio Alves,
Burgess Danielle,
Shema Reut,
Motlekar Nuzhat,
Napper Andrew D.,
Diamond Scott L.,
Pavitt Graham D.
Publication year - 2008
Publication title -
yeast
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.923
H-Index - 102
eISSN - 1097-0061
pISSN - 0749-503X
DOI - 10.1002/yea.1570
Subject(s) - biology , saccharomyces cerevisiae , yeast , plasmid , high throughput screening , escherichia coli , reporter gene , computational biology , cell , microbiology and biotechnology , biochemistry , gene , gene expression
Reporter‐gene assays that employ the Escherichia coli lacZ gene are ubiquitously employed in biological research. However, we were not able to readily identify a quantitative method that worked reliably with yeast ( Saccharomyces cerevisiae ) cells and that was compatible with high‐throughput screening and robotic liquid handling tools. We have therefore adapted a commercially available assay employing a 6‐ O ‐β‐galactopyranosyl–luciferin substrate to provide the required sensitivity with minimal sample handling times. Our assay uses only one‐tenth of the reagents suggested by the reagent manufacturer (Promega) for equivalent assays with mammalian cell cultures and produces rapid, sensitive and reproducible analysis with as little as 1 µl yeast cell culture and with < 100 cells. We demonstrate that the assay is compatible with yeast strains generated by the systematic yeast deletion project and functions equally well with genomically integrated or plasmid‐encoded lacZ reporters and with cells grown in complex or defined media. The high‐sensitivity, miniaturized format reduced sample handling required will make this assay useful for a wide range of applications. Copyright © 2007 John Wiley & Sons, Ltd.