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Star Republic: Guide for Biologists

Chloramphenicol Acetyl Transferase (CAT) Assay

1. Seed cells in 60 mm or 100 mm culture dish.

2. Transfect cells with CAT-report plasmid.

3. 36-48 hours after transfection. Remove the growth medium.

4. Wash cells 3 times with PBS.

5. Add 1 ml of TEN buffer (40 mM Tris-HCl pH 7.5, 1 mM EDTA, 150 mM NaCl).

6. Incubate at room temperature for 5 minutes.

7. Scrape cells using a cell scraper.

8. Transfer cells to a microcentrifuge tube.

9. Centrifuge at maximum speed to pellet cells.

10. Resuspend the pellet in 100-200 ml of 0.25 M Tris-HCl pH 8.0.

11. Freeze in dry ice/ethanol bath, thaw quickly in 37 C water bath, and vortex.

12. Repeat freeze-thaw two more times.

13. Heat inactivate endogenous deacetylase at 60 C for 10 minutes.

14. Centrifuge at maximum speed for 2 minutes.

15. Transfer supernatant to a fresh tube.

16. Store at -70 C or proceed to CAT activity assay.