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        CAT Assay

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        <center> <font>CAT Assay<br /> </font></center>
        Ref. Seed and Sheen, Gene 67, 271-277 (1988)

        Normal reaction is Chloramphenicol + Acetyl-CoA -> Acetylchloramphenicol

        Assay uses butyryl-CoA (more hydrophobic)

        14C-Cm+ Bu-CoA->Bu-14C-Cm Km (Cm) 10 µM
        Km (Ac-CoA) 50µM

        The 14C- Butyrylated Cm is separated from 14C Cm by phase extraction.

        ASSAY

        1. Make up cocktail, keep on ice; does 10 assays

        0.25 M Tris-Cl pH 8.0 0.7 ml (0.07/assay)
        14C Cm (57µCi/µmol; 25µCi/ml) 0.1 ml (10µl/assay)
        Bu CoA (5mg/ml) 0.2 ml (20µM/assay)

        Total 1.0 ml (100µl/assay)

        for assays longer than 1 hr, may need to add extra Bu-CoA to reactions at 1 hr.

        Reaction (set up on ice)

        Extract (50µl of 200 total) 50 µl
        Cocktail 100 µl
        Total 150 µl

        Incubate 37oC, 30 min to 5 hr to overnight (1 hr standard)

        To samples at room temp. add 300µl (2vols.) xylene; vortex hard 30 sec
        microcentrifuge 3 mins; remove 270 µl (care)
        add 150µl TE, vortex hard , spin 1 min, remove all xylene
        add 150µl TE, vortex hard, spin 1 min, remove 250µl xylene to a scintillation vial. Add 4 ml scint, count use

        1 CAT unit acetylates 1 µmol chloramphenicol/min 37oC

         

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