1. Wash cells transfected with beta-galactosidase reporter gene with PBS.
2. Add 100 ml of lysis buffer (25 mM Tris-phosphate,
pH 7.8, 2 mM DTT, 2 mM CDTA, 10% glycerol, 0.5% Triton X-100).
3. Incubate at room temperature for 20 minutes.
4. Transfer 30 ml of the cell extract to a new
96-well plate.
5. Add 150 ml CPRG buffer (60 mM Na2HPO4,
10 mM KCl, 1 mM MgSO4, 50 mM b-mercaptoethanol)
and 20 ml CPRG solution (4 mg/ml in H2O).
6. Incubate at 37 °C for 30 minutes or until the color has changed from yellow to dark red.
7. Stop reaction by adding 100 ml 1 M sodium carbonate and
0.05% ml Antifoam A.
8. Read absorbance at 575 nm with a plate reader.
9. Alternatively use the fluorogenic substrate 4-methylumbelliferone
ß-D-galactopyranoside (MUG) instead of CPRG and read with a fluorometer.
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