ASSAY FOR CHLORAMPHENICOL ACETYLTRANSFERASE
PREPARATION OF EXTRACTS
THIN-LAYER CHROMATOGRAPHY
1. Resuspend the cell pellet in 0.25 M TrisCl (pH 7.8).
2. Disrupt the cells.
3. Transfer the supernatant to a fresh microfuge tube.
4. Incubate the 50㎕aliquot of the extract for 10minutes at 65℃ to inactivate deacetylase.
5. Prepare CAT reaction mixture 1.
6. Mix each of the samples to be assayed with 80 ㎕ of CAT reaction mixture 1, and incubate the reactions at 37℃.
7. Add 1 ml of ethyl acetate to each sample, and mix thoroughly by vortexing. Centrifuge the mixtures at 12,000g for 5 minutes.
8. Transfer 900 ㎕of the upper phase to a fresh tube.
9. Evaporate the ethyl acetate under vacuum by placing the tubes in a rotating evaporator.
10. Redissolve the reaction products in 25 ㎕of ethyl acetate.
11. Apply 10-15 ㎕ of the dissolved reaction products to the origin of a 25 mm silica gel, TLC plate. (Mark the origin with a soft-lead pencil.) Apply 5 ㎕ at a time, and evaporate the sample to dryness with a hair dryer after each application.
12. Prepare a TLC chamber containing chloroform:methanol (95:5).
13. Place the TLC plate in the chromatography chamber, close the chamber, and allow the solvent to move.
14. Remove the TLC plate and allow it to dry. Place on the TLC plate adhesive dot labels marked with radioactive ink to align the plate with the film, and then expose the plate to X-ray film.
15. Develop the X-ray film and align it with the plate. Usally, three radioactive spots are visible. Nonacetylated, slow. Two monoacetylated, faster. Diacetylated, the fast.
16. To quantitate CAT activity, cut the radioactive spots from the TLC plate and measure the amount of radioactivity they contain in a liquid scintillation counter.