A rapid permeabilization procedure for accurate quantitative determination of β-galactosidase activity in yeast cells

Abstract

A procedure is described which allows the rapid permeabilization of yeast cells, Schizosaccharomyces pombe and Saccharomyces cerevisiae, for quantitative in situ assays of beta-galactosidase activity. Yeast cells are permeabilized by incubation in buffer containing 0.2% of the detergent sodium lauroyl sarcosinate without any need for washing or vortexing. This procedure is equally applicable to fresh and frozen samples. It is compared to earlier reported methods and found to be superior by being more accurate and less time-consuming.

Association of Bovine Papillomavirus Type 1 E6 oncoprotein with the focal adhesion protein paxillin through a conserved protein interaction motif

“…The resulting diploids were isolated on selective plates and individually grown in liquid glucose selective media for 4 h prior to spotting of the yeast cultures onto XGAL-containing galactose plates (the B42 fusion plasmid is induced by galactose), for a graphic indication of the 2-hybrid associations. Quantitative lacZ assays were as described in the Materials and methods and in (Kippert, 1995). The results shown are the lacZ activity relative to that obtained with wild-type lexA-BE6 fusion.…”

“…The plates were then read with a 96 well spectrophotometer at 650 nM to determine the yeast concentration in each well, and the plates subsequently re-centrifuged and the water discarded. The yeast were then re-suspended in 0.1 ml per well of beta galactosidase assay bu er (Z bu er) containing ONPG and 0.2% sarkosyl to permeabilize the yeast (Kippert, 1995). After the development of yellow color, the reaction was stopped with 0.1 ml of 0.1 M sodium carbonate, the plates were re-centrifuged, and 0.1 ml per well removed to a new microtiter plate and the plate optical density read at 410 nM.…”

Identification of inhibitors of yeast-to-hyphae transition in Candida albicans by a reporter screening assay

“…Reaction times for cell lysis and β-galactosidase reaction were chosen in accordance to previously published protocols (Kippert, 1995;Menzel, 1989;Miller, 1972) and to allow convenient handling time and parallel performance of large numbers of assays. The hydrolysis of ONPG in solvent controls remained proportional to the reaction time ( fig S1 D), demonstrating that the reaction was not yet saturated after 1.5 h.…”

Expression of Pseudomonas syringae type III effectors in yeast under stress conditions reveals that HopX1 attenuates activation of the high osmolarity glycerol MAP kinase pathway

“…Cultures were washed, diluted and grown in 4 ml selective medium containing galactose (2 %) and raffinose (1 %) to OD 600 0. then split into two tubes with 1 ml fresh selective medium containing galactose (2 %) and raffinose (1 %) with or without the respective stressing agent. To activate the HOG pathway, 0.5 M NaCl or 1 M sorbitol was added to the inducing medium for 1 h; to activate the CWI pathway, 7 mM caffeine was added to the inducing medium for 4 h; to activate the pheromone pathway, 5 mM a-factor was added to the inducing medium for 2 h; to induce the UPR pathway, 2 mM DTT was added to the inducing medium for 4 h. Cells were then collected and subjected to a b-galactosidase activity assay using 0.2 % sodium lauroyl sarcosinate as the permeabilizing agent (Kippert, 1995).…”