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MOLECULAR BIOLOGY: WORKING WITH PROTEINS

ENZYMATIC ASSAY: CAT

CHLORAMPHENICOL ACETYL TRANSFERASE (CAT) ASSAY

Chloramphenicol Acetyl Transferase (CAT) Assay Title | Procedure | Solutions | BioChemicals | Hints | Printable Version

Procedure Title | Procedure | Solutions | BioChemicals | Hints | Printable Version

A. Harvesting Cells 1. Wash a monolayer of cells three times using PBS Buffer. 2. Add between 0.5 to 1.5 ml of TBS Buffer and mix gently. 3. Incubate at room temperature for 5 min. 4. Scrape the cells to dislodge the cells from the culture plate and transfer the cell/TBS slurry to a 1.5 ml microcentrifuge tube. 5. Rinse the culture plate with an additional 0.5 ml of TBS Buffer and add to the microcentrifuge containing the cell/TBS slurry. 6. Centrifuge in a microcentrifuge for 2 min at setting "10" for 2 min. 7. Discard the supernatant and resuspend the cell pellet in between 100 μl to 300 μl of 0.25 M Tris-Cl, pH 7.5. 8. Freeze the cells in a Dry Ice 100% Ethanol Bath for two minutes. 9. Thaw the cell in a water bath at 37°C. 10. Repeat the freeze-thaw procedure two more times (Steps #8 and #9). 11. Centrifuge the thawed cell slurry in a microcentrifuge tube at setting "10" for 5 min. 12. Transfer the supernatant to a new 0.5 ml microcentrifuge tube (see Hint #2). 13. Determine the protein concentration of the cell supernatant (see protocol on Protein Quantification) or determine the activity of beta-galactosidase (see protocol on Beta-Galactosidase Quantification). B. Chloramphenicol (CAT) Assay 1. Prior to starting assay, determine the total number of samples (remember that there are three samples per unknown). Label all tubes accordingly and premix the necessary volume of the following reagents. CAT Assay Solution for 10 assays: 1 ml of ddH2O 625 μl of 4X Assay Buffer 200 μl of 5 mM Acetyl CoA 75 μl of 0.1 M ATP 7.5 μl of Acetyl CoA Synthetase 4.2 μl of [3H]-Acetic Acid 100 μl of 10 mM Sodium Acetate 5 μl of 0.5 M Chloramphenicol 2. Aliquot between 25 to 50 μl of cell extract (prepared in Section A) and 2.0, 2.7 or 8.0 μl of 0.5 M Chloramphenicol to each assay tube. 3. Add 200 μl of CAT Assay Solution (prepared in Step #1) and mix completely. 4. Incubate for 45 to 60 min at 37°C (see Hint #3). 5. To stop the reaction add 1 ml of Benzene (CAUTION! see Hint #1 and Hint #4). 6. Vortex each tube for 30 seconds. 7. Centrifuge in a microcentrifuge at maximum speed for 1 min. 8. Carefully remove 800 μl of the upper phase (Benzene phase) and place into properly labeled scintillation vials. 9. Allow the Benzene to air dry (this will take approximately 4 hours). 10. Add 3 ml of Scintillation Fluid and count the radioactivity. 11. Calculate the amount of CAT activity.

Solutions Title | Procedure | Solutions | BioChemicals | Hints | Printable Version

10 mM Sodium Acetate    Store at room temperature Acetyl CoA Synthetase    in PBS Aliquot and store at -20°C 5 mg/ml S-Acetyl Coenzyme A Synthetase (Boehringer) 5 mM Acetyl CoA    in 2.6 ml ddH2O 10 mg Acetyl CoA Solution is stable for approximately 2 weeks Aliquot and store at -20°C 0.1 M ATP    Store at -70°C 60 mg ATP in 0.8 ml ddH2O Adjust final volume to 1 ml Adjust pH to 7.0 using 0.1 M NaOH 0.1 M NaOH 0.5 M Chloramphenicol    100 mg Chloramphenicol in 1 ml of 100% Ethanol 0.25 M Tris, pH 7.5    Store at room temperature in Sterile ddH2O TES Buffer    Store at room temperature 40 mM Tris-Cl, pH 7.5 Autoclave to sterilize 1 mM EDTA 150 mM NaCl TBS Buffer    Store at room temperature 2.7 mM KCl 10 mM Tris-Cl 137 mM NaCl pH 7.5 PBS Buffer    Store at room temperature 1.5 mM Potassium Phosphate Monobasic (KH2PO4) 2.7 mM KCl 137 mM NaCl 10 mM Sodium Phosphate Dibasic (Na2HPO4) pH 7.5 Assay Buffer (4X)    Store at room temperature 400 mM Tris-Cl, pH 7.5 Autoclave to sterilize 300 mM KCl 24 mM MgCl2 [3H]-Acetic Acid    6 mCi/mmol [3H]-Acetic Acid (CAUTION! see Hint #1)

BioReagents and Chemicals Title | Procedure | Solutions | BioChemicals | Hints | Printable Version

Tris-Cl S-Acetyl Coenzyme A Synthetase Chloramphenicol Benzene [3H]-Acetic Acid ATP Tris Potassium Phosphate, Monobasic Ethanol Sodium Phosphate, Dibasic Acetyl CoA Potassium Chloride Sodium Chloride Sodium Acetate Scintillation Fluid Magnesium Chloride Sodium Hydroxide

Protocol Hints Title | Procedure | Solutions | BioChemicals | Hints | Printable Version

1. CAUTION! This substance is a biohazard. Consult this agent's MSDS for proper handling instructions. 2. If not used immediately, the cell supernatant can be stored at -20°C. 3. The following control solutions should always be prepared when performing a CAT Assay: buffer alone (indicates background), nontransfected cell extract (negative control) and Acetyl Coenzyme A transferase (positive control). 4. Perform the remaining steps of this procedure in a chemical fume hood.

   

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