1. Remove culture media and wash twice with PBS.
2. Scrape cells into PBS, transfer to a centrifuge tube.
3. Spin down cells at 250x g for 5 minutes. Remove the supernatant.
4. Resuspend the pellet in cold 0.25 M Tris-HCl pH8.0. For cells
transfected with beta-galactosidase reporter gene, use 100-1000
ml of Tris-HCl pH8.0 per 60 mm culture dish.
5. Subject the extract to 3 freeze/thaw cycles by freezing the extract
in liquid nitrogen or dry ice/ethanol bath and thawing quickly in a 37
°C water bath. Vortex the extract vigorously after each thaw cycle.
6. Centrifuge the extract at 12,000 rpm for 5 minutes in a microcentrifuge
at 4 °C.
7. Transfer the supernatant to a fresh eppendorf tube, store at -70 °C or put
on ice for immediate activity assay.
8. Add to a fresh tube 10 ml extract, adjust
volume to 150 ml with 0.25 M Tris-HCl pH8.0.
More extract can be used if the beta-galactosidase activity is low. Also
prepare a control tube with 150 ml 0.25 M Tris-HCl pH8.0.
9. Add 150 ml of 2X Assay Buffer (200 mM NaPO
4 buffer pH7.3, 2mM MgCl2,
100 mM beta-mercaptoethanol, 1.33 mg/ml ONPG) to the extract and the control.
If a precipitate is present in the 2X Assay Buffer, warm briefly in a 37 °C
water bath before use.
10. Incubate at 37 °C from 30 minutes to overnight until a faint yellow color has developed in the
sample but not in the control.
11. Stop the reaction by adding 500 ml of 1 M
sodium carbonate. Prewarm sodium carbonate to 37 °C to dissolve the crystals.
12. Read the absorbence at 420 nm, using the control as the blank.