Chloramphenicol Acetyl Transferase Assay

1. Tissue extracts are prepared by homogenization in 0.2 mL of 0.25M Tris-HCl, pH 7.8, followed by three rounds of freeze-thawing by immersion in liquid nitrogen (3 min) and in a 37°C water bath (3 min). Vortex the suspension thoroughly after the final thawing step, and then spin at 14,000 rpm in a microcentrifuge at 4°C for 10 min. Retain the supernatant for the analysis of CAT activity. Prior to assay, heat the extract to 65°C for 10 min to destroy endogenous CAT inhibitors {see Note 1).

2 Set up the incubation mixture as follows:

20 pL 8 mM chloramphenicol 30 pL tissue extract 20 pL diluted acetyl CoA solution 30 pL 0.25M Tris-HCl, pH 7.8, and 5 mM EDTA. The diluted acetyl CoA solution is prepared by diluting a 0.02-mL aliquot of 14C acetyl CoA containing 1 mCi with 0.5 mM unlabeled acetyl CoA to a concentration of 5 mCi/mL.

3. Incubate at 37°C for 1 h, and then transfer the samples to an ice bath.

4. Extract the labeled reaction products twice into 100-pL aliquots of cold ethyl acetate. After addition of ethyl acetate to each sample, mix the layers by vortexing, and then separate by centrifugation at 14,000 rpm for 3 min at 4°C. After the first extraction, remove 80 pL of the orgamc phase; after the second extraction, remove 100 pL of the organic phase and combine the two.

5. Add the combined organic extracts to 2 mL of scintillation fluid, and determine radioactivity by liquid scintillation spectroscopy.

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