A Saccharomyces cerevisiae cell-based quantitative beta-galactosidase assay compatible with robotic handling and high-throughput screening.
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-beta-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 microl 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.
de Almeida
Rogerio Alves
RA
Faculty of Life Sciences, University of Manchester, Manchester M13 9PT, UK.
Burgess
Danielle
D
Shema
Reut
R
Motlekar
Nuzhat
N
Napper
Andrew D
AD
Diamond
Scott L
SL
Pavitt
Graham D
GD
D050976:Basic-Leucine Zipper Transcription Factors; D004268:DNA-Binding Proteins / Q000235:genetics; D017930:Genes, Reporter; D007763:Lac Operon; D008156:Luciferases / Q000235:genetics; D008164:Luminescent Proteins / Q000032:analysis; D012371:Robotics / Q000379:methods*; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics; D014157:Transcription Factors / Q000235:genetics; D001616:beta-Galactosidase / Q000032:analysis*
D050976:Basic-Leucine Zipper Transcription Factors; D004268:DNA-Binding Proteins; C501391:GCN4 protein, S cerevisiae; D008164:Luminescent Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D008156:Luciferases; D001616:beta-Galactosidase