Assay for chloramphenicol acetyl transferase in amoebal lysates

Tim Burland (Last Update May 4, 1993)

1. Make a lysate of 3-5x10⁷ amoebae:

For amoebae growing on plates:

• Flood plates with BSS and scrape amoebae off surface with a sterile glass spreader.
• Recover cells from plates with pipette.
• Continue as for amoebae growing in broth.

For amoebae growing in broth:

• Spin down for 2 min at 200 x g, then resuspend in 1mL BSS
• Transfer to 1.5-mL microfuge tube containing 100mg 500um acid-washed glass beads (Sigma #9268)
• Spin down 25 sec. in microfuge @ 7000rpm
• Aspirate supernatant with a Pasteur pipette
• Add 100uL 250mM Tris pH 7.8 to pellet, and vortex full speed 30sec.
• Quick-freeze in dry ice / EtOH bath.

2. Inactivate background acetyl transferases

• Thaw Lysates at 37^o
• Mix together:  0-100uL cell lysate (or 0.05units purified CAT ((Sigma)) for +ve control), and 250mM Tris pH7.8 to 155uL
• Incubate 60^o for 15min.

3. Enzyme reaction:

To the heat-inactivated extracts, add:

• 5ul ¹⁴C-Chloramphenicol (0.125uCi; 50mCi/mmol; Amersham CFA.754)
• 20uL 4mM Acetyl CoA (add 1mg AcCoA to 333uL 250mM Tris immediately before use)  (You can mix the acetyl CoA and chloramphenicol together just before adding to reactions).
• Incubate 37^o 60min.

4. Extract and separate chloramphenicols:

• Add 1ml ethyl acetate containing 20ug/mL unlabelled chloramphenicol.
• Vortex each reaction 3x5sec.
• Spin 30sec microfuge
• Take off upper organic phase and put in clean microfuge tube
• Dry off all ethyl acetate under vacuum
• Add 15-20uL ethyl acetate (containing 100ug/mL chloramphenicol) to dissolve pellet.
• Spot solution onto a 20x20cm 250um silica-gel TLC plate (Baker Si250, Cat #7000-04)
• Resolve acetylated chloramphenicols by ascending chromatography in 95% chloroform / 5% methanol.
• Dry plate in air and expose to XAR-5 X-ray film overnight or longer.