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Star Republic: Guide for Biologists

Chloramphenicol Acetyl Transferase (CAT)

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.

5. Vortex.

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.

5a. Vortex.

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 1 hour.

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 scintillation counting.

See also Chloramphenicol Acetyl Transferase (CAT) Assay.