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Star Republic: Guide for Biologists

ONPG beta-galactosidase activity assay

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.