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Star Republic: Guide for Biologists

beta-galactosidase activity assay --- 96-well

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 -D-galactopyranoside (MUG) instead of CPRG and read with a fluorometer.