In this protocol, we list three different assays that can be used to determine CATase activity expressed after transfection of mammalian cells with the reporter gene vector pCAT3 (please see Figure 17-3). This experiment was derived from the next volume of the Guide to Molecular Cloning Experiments (Third Edition) by [American] J. Sambrook D.W. Russell.
Operation method
Determination of chloramphenicol acyltransferase content by thin-layer chromatography experiment
Materials and Instruments
Cultured mammalian cells transfected with the pCAT vector carrying the DNA of interest Move makings For more product details, please visit Aladdin Scientific website.
CAT Reaction Mixture 1 Ethyl Acetic Acid Lysis Buffer Phosphate Buffer without Calcium and Magnesium Salts Thin Layer Chromatography (TLC) Solvents Tris-Cl Radioactive Ink
Dry ice Ethanol bath Marker for ethanol-insoluble inks Rotary vacuum evaporator Rubber mallet Thin layer chromatography plate Thin layer chromatography bath Water bath preset at 65°C
Buffers and solutions
See Appendix 1 for components of storage solutions, buffers and reagents.
Dilute the storage solution to the appropriate concentration.
CAT Reaction Mixture 1
1mol/LTris-Cl (pH 7.8) 50ul
[14C ] Chloramphenicol (60mCi/mmol, diluted to 0.1mCi/ml with water) 10ul
Acetyl Coenzyme A (freshly prepared, aqueous concentration 3.5 mg/ml) 20ul
For each 50ul of cell lysate assayed, 80ul of CAT reaction mixture was prepared. The reaction mixture is prepared just before use.
Acetylase A is the S-acetylated form of coenzyme A. Two iodine units are derived from lipids in this form. Two iodine units enter the citric acid cycle from fats and carbohydrates in this form. Acetyl Coenzyme A serves as a cofactor for acetylation in many biological reactions, such as the acylation of chloramphenicol by CAT in this protocol.
Ethylacetic acid
Ethylacetic acid ( CH2COOCH2CH3 ) is an organic solvent used in the thin layer chromatographic determination of CAT activity.1 part ethylacetic acid mixed with 19 parts chloroform is the solvent that separates the acetylated and non-acetylated forms of chloramphenicol on silica gel plates. Ethylacetic acid is a very flammable (flash point = -4°C) volatile ester with the odor of ripe fruit. 
Lysis Buffer
0.1 mol/L Tris-Cl (pH 7.8)
0.5% (V/V) TritonX-100
Used when lysing cells by decontamination (see step 4).
The same lysis buffer can be used to prepare cell extracts for measuring β-galactosidase activity (please see Scheme 7).
Important: Cell Lysis Buffer should be made from high purity TritonX-100. Lower grade detergents will inhibit CATase activity. TritonX-100 can be replaced by NonidetP-40 with a detergent of 0.125% (V/V) in the lysis buffer.
Phosphate Buffer Solution (PBS) without Calcium and Magnesium Salts
Thin Layer Chromatography (TLC) Solvent
190 ml chloroform
10 ml Methanol
Each TLC tank requires 200 ml of preparation. two TLC plates can be used in each tank.
Tris-Cl (1mol/L, pH 7.8)
Radioactive compounds
Radioactive ink
Used for spot marking to indicate the direction of the radiographic autoradiograph of the TCL cabinet.
Special equipment
Dry ice/ethanol bath
For lysis of cells by freeze-thaw method.
Markers using ethanol-insoluble ink
Rotary vacuum evaporator
Examples include the Savant Speed Vac brand.
Rubber rods
Thin layer chromatography plates (e.g. Sybron SIL G/UV254, Brinkmann)
Thin layer chromatography bath (27.5 cm X 27.5 cm X 7.5 cm)
These tanks are available from scientific supply stores (e.g. Sigma-Aldrich Techware).
Water bath preset at 65°C
Cells and tissues
Cultured mammalian cells transfected with pCAT vector carrying DNA of interest
Cells need to be cotransfected (using one of the transfection protocols in Chapter 16) with a CAT reporter molecular vector (e.g., the pCAT3 series) and a plasmid carrying the β-galactosidase gene (pCMV-SPORT-β-gal; see Figure 16-2 in Chapter 16) or another reporter gene suitable for normalizing CAT measurements. 3.
Methods
Preparation of Transfected Cell Precipitates
1. Gently aspirate the culture medium from monolayers of transfected cells grown in 90 mm tissue culture dishes. The monolayers were washed 3 times with 5 ml of PBS free of calcium and magnesium salts.
2. Place the dish at an angle for 2-3 minutes so that the last remaining PBS flows to one side. Aspirate off the PBS residue. Add lml PBS to each petri dish and scrape the cells into the centrifuge tube with a rubber mallet. Ice bath until all petri dishes are processed.
3. Collect the cells by centrifugation at maximum speed for 10s at room temperature. Gently resuspend the cell precipitate with lml ice-cold PBS and then centrifuge again to collect the cells. Remove the cell sediment and PBS residue from the walls of the tube. The cell precipitate can be stored at -20°C for future analysis or a cell extract can be prepared using one of the methods in step 4.
PBS is easily removed with a disposable pipette attached to a vacuum tube. touch the pipette tip to the surface of the liquid and aspirate slowly. As the liquid is falling, keep the tip as far away from the cell sink as possible. Keep the pipette tip as far away from the cell deposit as possible and then use the tip to suck off any droplets that have adhered to the walls of the tube.
Preparation of Cell Extracts
4. Cells are lysed either by repeated freeze-thawing or by treating the cells with a buffer containing a decontaminant. The latter method is quick and easy and is suitable for measuring CAT, β-galactosidase and other marker genes in 96-well plates (Scheme 6) (Bignon et al. 1993).
Cell lysis by repeated freeze-thawing
a. Resuspend cell precipitates with l00ul of 0.25mol/L Tris-Cl (pH 7.8) in a 90 mm Petri dish. Shake vigorously to break up the cell clumps.
b. Cells are frozen in a dry ice/ethanol bath, melted at 37°C, and subjected to 3 cycles to disrupt the cells. Make sure that the tubes have been previously labeled with ethanol-insoluble ink.
The cells should be centrifuged at maximum speed for 5 min at 4°C. c. Disrupted cells should be centrifuged at maximum speed for 5 min at 4°C. The supernatant should be transferred to a new centrifuge tube. Take 50ul of the supernatant for CAT measurement and store the remaining extract at -20°C.
Cells are lysed with buffer containing detergent.
a. Resuspend the cell precipitate from step 3 with 500ul of Lysis Buffer. Incubate the mixture at 37°C for 15 min.
Cells grown in 35 mm culture blood require 100ul of Lysis Buffer for extraction.
b. Remove cell debris by centrifugation at maximum speed for 10 min. Recover the supernatant. One of the methods described in this protocol is chosen to measure CAT activity. The remaining clear lysate is snap-frozen in liquid nitrogen and stored at -70°C.
Determination of CAT Activity by Thin Layer Chromatography
5.50ul of cell extract is incubated at 65°C for 10 min to inactivate the endogenous deacetylase. If the extract is cloudy and opaque at this point, centrifuge at 4°C for 2 min at maximum speed to remove particles.
6. Mix each sample to be tested with 80ul of CAT Reaction Mix 1 and incubate at 37°C. Incubate for as long as necessary. The length of incubation time is related to the concentration of CAT in the cell extract, which in turn is related to the strength of the promoter and the cell type under study. In most cases, incubation for 30 min to 2 h is sufficient.
If the expression of the CAT gene in the transfected cells is very low, the reaction can be incubated for a longer period of time (up to 16 h). However, in most cases, it is recommended to add 10ul of Acetyl Coenzyme A after 2 h of incubation for each reaction.
7. Add lml of acetoacetate to each sample and shake the solution 3 times for 10s each time to thoroughly mix the solution. The mixture is centrifuged at maximum speed for 5 min at room temperature.
The acetylated chloramphenicol goes into the organic phase (upper layer); the non-acetylated chloramphenicol remains in the aqueous phase.
8. Transfer 900ul of the organic phase to a new tube using a pipette, being careful to avoid mixing the aqueous and intermediate phases. Discard the tube containing the lower phase as radioactive waste.
9. Place the tube in a rotary evaporator (e.g. Savant SpeedVac brand) and allow the ethylacetic acid to evaporate under vacuum for about lh.
10. Add 25ul of ethylacetic acid to each tube and shake gently to dissolve the reaction products.
11. Add 10-15ul of dissolved reaction product to the starting point of a 25 mm silica gel TLC plate. Mark the starting point on the plate with a soft graphite pencil. Add 5ul of sample at a time and blow the sample dry with a hair dryer.
12. Prepare a TCL bath containing 200 ml of TCL solvent. Place the TCL plate in the tank, close the container, and move the solvent front to approximately 75% of the top of the plate.
Many different TCL plates and fingerlings can be used to separate acetylated chloramphenicol. We usually use chloroform:methanol (95:5) and Sybron SILG/UV254 (Brinkmann Corp.) TCL plates.The TCL method and its difficulties are well described and illustrated by Touchstone (1992).
13. Remove the TCL plate from the bath and dry it at room temperature. Dot-mark the TCL plate with radioactive ink to easily align the plate with the film, and then expose the plate to X-ray film. Alternatively, the plate is placed in a box for phosphor screen analysis. The box is left at room temperature for an appropriate period of time.
The TCL plate should not be covered with Satan film because the film will cut off the weaker radiation from the 14C isotope.
14. Rinse the X-ray film and align the position with the TCL plate. In addition, the image plate of the phosphor screen analyzer can be exposed with a chromatogram or the TCL plate can be taken for scanning.
Three radioactive spots are usually visible. The spot that migrates the closest distance from the starting point is the non-acetylated chloramphenicol that enters the ethyl acetate. The other two spots that migrate slightly faster are chloramphenicol that has been acetylated at one of the two potential acylation sites. The third spot that migrates more rapidly, which is bis-acetylated chloramphenicol, is seen only after prolonged incubation with high concentrations of CAT or with large amounts of extracted protein.
15. In order to quantify CAT activity, the radioactive spot needs to be cut out of the TCL plate and the amount of radioactivity measured using a liquid flash counter (please see Appendix 9). Take another cell extract (from step 3 above) and determine the protein concentration in the extract using a rapid colorimetric method such as the Bradford assay. Before determining the protein concentration, dilute the Triton X-100 to ≤0.1% to avoid cross-reactivity.CAT activity is expressed as the number of picomoles of acetylated product produced per unit of time per milligram of cell extract protein.
The method of silica gel thin-layer dialysis followed by gel cutting and scintillation counting of modified chloramphenicol products becomes tedious when working with large numbers of samples, and an alternative method of product quantification in these cases is to scan the TCL plate with a phosphor-screen device.
Chloramphenicol hexanoylation that remains 'intrinsic' in the absence of cell extracts may form a background. If this will be a problem, try reducing the concentration of chloramphenicol in the experiment by a factor of 2-4 (Heard et al. 1987). 
