Beta galactosidase enzymatic activity assay for yeast
Method Article
Beta-galactosidase activity assay for yeast
https://doi.org/10.1038/protex.2019.002
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Beta galactosidase enzymatic activity assay for yeast
Z Buffer: 60 mM Na2HPO4, 40 mM NaH2PO4·H2O, 10 mM KCl, 1 mM MgSO4, pH 7
β-mercaptoethanol ( 161-0710, Bio-Rad)
Zymolyase-20T (120491, SEIKAGAKU CORPORATION)
Protein Assay Dye Reagent Concentrate (500-0006, BIO-RAD) for Bradford Assay
ONPG (N1127, SIGMA ALDRICH)
Collect approximately 5 × 107 cells from yeast exponentially growing cultures
Pellet cells by centrifugation at 4000xg at 4ºC and wash once with water
Cells can be stored at -20ºC
Resuspend cells with 200 μl of Z Buffer + β-mercaptoethanol (0.27% (v/v)) containing 1mg/mL zymolyase-20T
Incubate samples 20-30 min at 30ºC to digest cells
Centrifuge cells at 13400xg for 5 min at 4°C
Transfer the supernatant to a new eppendorf
Use 1 or 2 μL of the extract to quantify the amount of protein using the Bradford assay (Concentration)
Mix 180 μl of extract (Volume) with 700 μL of Z Buffer + β-mercaptoethanol
Start the reaction by adding 200 μL of 4mg/mL ONPG (o-nitrophenyl- β-D-galactopiranoside
Incubate samples at 30ºC until they become yellow coloured (up to 90 minutes)
When yellow colour appears (Time), stop the reaction with 500 μL of Na2CO3 1M
Centrifuge cells at 13400xg for 1 min to pellet cells
Measure A460
β -galactosidase activity is expressed in arbitrary units (U), from the following formula:
U=(A460 x1000)/(Time (〖min) x Volume (µL)x Concentration (µg/µL)〗 )
The authors declare no competing financial interests
This protocol has been posted on Protocol Exchange, an open repository of community-contributed protocols sponsored by Nature Portfolio. These protocols are posted directly on the Protocol Exchange by authors and are made freely available to the scientific community for use and comment.
posted
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