A rapid method for liquid β-galactosidase reporter assay in Saccharomyces cerevisiae

Although β-galactosidase assay is widely used for various studies in yeast a quantitative estimation of low enzyme activities with standard reactives remains hampered. It requires long reaction time and large amounts of cells. To overcome existing limitations we developed protocol, which incorporates realization reaction in miniaturized format, cell lysis in reaction buffer and simplification the normalization of β-galactosidase activity. These features allow faster reaction kinetics, accurate and simple quantification of low enzyme activities. To perform studies in vivo conditions we constructed a reporter plasmids based on the low copy yeast vector Ycp50. We adapted our assay on the yeast protein Rpn4 which is highly unstable with a half-life of only 2 min. We demonstrated that detection of Rpn4–LacZ fusion is achieved in 40 min in our method, whereas in standard assay it requires 4–5 h. Moreover, we implemented our approach for promoter dissection investigation. Thus, we present rapid, convenient and less labor-intensive method for assessment β-galactosidase activity.     

Rpn4p without the DNA-Binding Domain Provides Saccharomyces cerevisiae Resistance to Oxidative Stress and Cycloheximide

Molecular Biology - The ubiquitin-proteasome system is involved in the control of all essential molecular processes under normal conditions and the response of cells to stress. Rpn4p serves as a...