1. Cell extract in a suitable lysis buffer (e.g., (I) 10 mM Tris-HCl pH8.0,
1 mM EDTA, 150 mM NaCl, 0.5% NP-40 or (II) 250 mM Tris-HCl pH 7.8).
2. Setup 100 ml reactions. The final concentrations
are: 0.2 mM acetyl CoA, 2 mM chloramphenicol, 200 mM Tris-HCl pH 8.0,
and 50 mCi 14C
acetyl coenzyme A.
2a. Alternatively setup 100 ml reactions with
the final concentrations of 50 mCi 14C
chloramphenicol, 0.2 mM n-Butyryl CoA (or acetyl CoA) , 200 mM Tris-HCl pH 8.0.
3. Incubate at 37 °C for 60 minutes to overnight.
Liquid scintillation counting (LSC)
4. Add 2 volumes of xylene.
6. Centrifuge for 2 minutes at maximum speed to separate aqueous and organic phases.
7. Transfer the organic upper phase (xylene) to a fresh tube.
8. Add 1/2 volume of TE (or 0.25 M Tris-HCl pH 8.0) to the tube containing
xylene phase. Repeat steps 5-7.
9. Add 1/2 volume of TE (or 0.25 M Tris-HCl pH 8.0) to the tube containing
xylene phase. Repeat steps 5-6.
10. Transfer the organic upper phase (xylene) to a scintillation vial.
11. Add an appropriate amount of scintillation fluid and count.
Silica gel thin layer chromatography (TLC)
4a. Add 2 volumes of ethyl acetate.
6a. Centrifuge for 2 minutes at maximum speed to separate aqueous and organic phases.
7a. Transfer the organic upper phase (ethyl acetate) to a fresh tube.
8a. Vacuum dry ethyl acetate.
9a. Resuspend in 15-30 ml of ethyl acetate.
10a. Spot 10 ml onto the origin of
a 20x20 cm silica-gel TLC plate.
11a. Pre-equilibrate a TLC developing chamber with chloroform/methanol (95:5) for
12a. Lower the TLC plate into the chamber, origins at the bottom.
13a. Run until the solvent has reached the top half of the plate.
14a. Air-dry the plate.
15a. Expose to X-ray film (with intensifying screen) or process using phosphorimage.
16a. The spots on TLC plates may be cut and quantitated d using liquid
See also Chloramphenicol Acetyl Transferase (CAT) Assay.