Activity Assay of Chloramphenical Acetyl Transferase CAT

Protocol 1. Preparation of a Cytoplasmic Extract from Transfected Cells for the CAT Assay

1. For transient transfection, approximately 48 to 72 h after transfection, remove the culture medium from the dish and wash the cells twice with 10 ml of PBS. For stable transfection, proteins can be extracted from stably transfected cells.

2. Aspirate the PBS buffer and add 1 ml of fresh PBS. Loosen and resuspend the cells into the buffer using a sterile rubber policeman.

3. Transfer the cell suspension into a fresh microcentrifuge tube, centrifuge at 1200 x g for 5 min and decant the supernatant.

Note: Do not centrifuge at top speed. Otherwise, the cells may rupture.

4. Resuspend the cells in 0.3 ml of 250 mM Tris-HCl, pH 8.0.

5. Lyse the cells by three to four cycles of freezing (dry ice or dry ice-methanol bath, 5 min) and thawing (37°C, 5 to 10 min).

6. Centrifuge at 10,000 x g for 10 min in a microcentrifuge and transfer the supernatant (extract) to a fresh tube. Store the extract at -70°C prior to use.

FIGURE 8.12 DNA analysis by Southern blotting. Lane 1: nontransfected cells. Lane 2 and lane 3: putatively transfected cell lines.

FIGURE 8.12 DNA analysis by Southern blotting. Lane 1: nontransfected cells. Lane 2 and lane 3: putatively transfected cell lines.

Protocol 2. Chloramphenicol Acetyl Transferase (CAT) Enzyme Assay The CAT enzyme, expressed by the CAT gene, originally comes from bacteria. It catalyzes the conversion of [14C]chloramphenicol to its 1-acetyl and 3-acetyl derivatives. The derived products are separated from the unconverted compound by thin-layer chromatography (TLC) on plates precoated with silica gel, which are then exposed to x-ray film for autoradiography.

1. Add the following components to a microcentrifuge tube: Prepared cell extract, 0.1 ml

500 mM Tris-HCl, pH 8.0, 60 ml [14C]chloramphenicol (0.025 mCi/ml), 10 ml n-Butyryl coenzyme A (5 mg/ml), 10 ml Total volume of 180 ml

Note: A standard curve for CAT activity includes one blank with the CAT enzyme.

2. Incubate reaction at 37°C for the optimum time period (0.5 to 20 h) as determined from prior experiments.

3. Terminate the reaction by adding 0.5 ml of ethyl acetate to the tube for the TLC assay and vortex for 1 min.

4. Centrifuge at 10,000 x g for 4 min and transfer the upper, organic phase to a fresh tube and dry down the ethyl acetate in a vacuum evaporator.

5. Resuspend the residue in 20 ml of ethyl acetate and spot 10 ml of each sample onto a silica gel TLC plate and air-dry.

6. Place the TLC plate in a tank containing 100 ml of solvent (chloro-form:methanol, 95:5). Once the solvents migrate up to about 1 cm from the top of the plate, remove the plate and air-dry.

7. Expose the plate to x-ray film for 10 h or overnight at room temperature. In order to carry out a quantitative assay, for each sample, slice a square corresponding to the monoacetylated form from the silica plate and place in a vial. Add 5 to 10 ml of scintillation fluid to the vial and count in a scintillation spectrometer.

Luciferase Assay

Luciferase is encoded by the luciferase gene. Its assay is based on an oxidation reaction mediated by luciferyl-CoA and light is produced as a result of this reaction.

1. For each sample, add 30 ml of the cell extract as described previously to a vial containing 150 ml of luciferase assay reagent at room temperature.

2. Quickly place the reaction mixture in a luminometer and measure light produced for a period of 10 s over a 2- to 3-min period, depending on the sensitivity.

Note: The luciferase activity decreases very rapidly with time.

Reagents Needed

Luciferase Assay Reagents 20 mM Tricine

2.67 mM MgSO4

33.3 mM DTT

0.27 mM Coenzyme A

0.47 mM Luciferin

ß-Galactosidase Assay

1. For each sample, add the following components to a microcentrifuge tube: Magnesium solution, 3 ml

CPRC (4 mg/ml), 66 ml 0.1 M Sodium phosphate buffer, 200 ml Cell extract, 31 ml Total volume of 300 ml For blank:

Magnesium solution, 3 ml

0.1 M Sodium phosphate buffer, 200 ml

Total volume of 300 ml

2. Incubate at 37°C for 30 to 60 min and add 0.7 ml of 1 M of Na2CO3 to terminate the reaction.

3. Measure the absorbency at A574 nm against blank reference.

Reagents Needed Magnesium Solution

0.1 M MgCl2 5 M 2-Mercaptoethanol

0.1 M Sodium Phosphate Buffer, pH 7.3 CPRG Solution

4 mg/ml Chlorophenol red-ß-D-galactopyranoside in 0.1 M sodium phosphate buffer

ß-Galactosidase Staining of Cells

This is a fast procedure to assay the activity of the lac Z gene if it is incorporated into the DNA construct. ß-galactosidase can hydrolyze X-gal (5-bromo-4-chloro-3-

indolyl-P-D-galactoside) and yield a blue precipitate, staining the cells blue. Protocol is as follows:

1. Culture the stably transfected cells in a Petri dish for 1 to 2 days.

2. Aspirate the medium and rinse the cells with PBS.

3. Fix the cells in 0.2% glutaraldehyde or 4% paraformaldehyde (PFA) for 5 to 10 min at room temperature.

5. Stain the cells with X-gal solution for 1 to 24 h at 37°C.

6. Wash the cells for 3 to 5 min in PBS. Any blue cells or colonies indicate that the cells express P-galactosidase and that they most likely contain the targeted gene.

Reagents Needed

Potassium Phosphate Buffer (PBS) 2.7 mM KCl 1.5 mM KH2PO4 135 mM NaCl 15 mM Na2HPO4 Adjust pH to 7.2, then autoclave.

Preparation of 4% (w/v) Paraformaldehyde (PFA) Fixative (1 l)

a. Carefully weigh out 40 g paraformaldehyde powder and add it to 600 ml preheated dd.H2O (55 to 60°C) and allow the paraformaldehyde to dissolve at the same temperature with stirring.

Caution: Paraformaldehyde is toxic and should be handled carefully. The preparation should be carried out in a fume hood on a heating plate. The temperature should not exceed 65°C and the fixative should not be overheated.

b. Add 1 drop of 2 N NaOH solution to clear the fixative.

c. Remove from heat source and add 333.3 ml (1/3 total volume) of 3X PBS (8.1 mM KCl, 4.5 mM KH2PO4, 411 mM NaCl, 45 mM Na2HPO4, pH 7.2, autoclaved).

d. Adjust the pH to 7.2 with 4 NHCl and bring the final volume to 1 l with dd.H2O.

e. Filter the solution to remove undissolved fine particles, cool to room temperature and store at 4°C until use.

X-Gal Solution

PBS solution contains 0.5 mg/ml X-gal and 10 mM K3[Fe(CN)6]. X-gal stock solution contains 50 mg/ml in dimethylsulfoxide or dimethylformamide. The stock solution should be kept in a brown bottle at -20°C prior to use.


Detailed protocols are described in Chapter 9.


Detailed protocols are given in Chapter 9.


1. Low efficiency of transfection of cells by liposomes, electroporation and calcium phosphate methods. If cells are normal and the cell density is appropriate, it is likely that the amount of DNA constructs used and parameters applied in transfection are not optimal. Make sure to optimize the conditions for transfection of the cells, including DNA concentration, lipids ratio, and voltage pulse. In case of stable transfection, ensure that the concentration of the drug used in the selection is not too high. A killing curve should be established.

2. Low titer of retroviruses. The virus stock is overdiluted or there is a low density of retrovirus particles due to low efficiency of stable transfection of packaging cells with modified virus DNA constructs. Try to use a good packaging cell line and optimize the parameters for transfection. Reinfection may be performed to amplify the virus titer.

3. Low efficiency of transfection with frozen high-titer viruses. Assuming transfection conditions are optimal, it is very likely that the viruses have lost significant activity. It is recommended that high-titer viruses be used in a couple of hours. Frozen aliquots should not be frozen and thawed.

4. Southern blot analysis shows that sense cDNA is correctly integrated into the genome of the host cell, but no exogenous mRNA and protein are expressed. Something is wrong with the inserted cDNA. This could be due to a minor mutation that occurs during integration. Once this occurs, the best way to deal with it is to forget it. However, if verifying the cell clone is desirable, a PCR approach may be used to sequence the insertion site, especially the 5' end of the cDNA introduced. If some bases are missing or ORF has been shifted, there is no way to rescue the cell clone.

5. Low-level expression of mRNA and protein of interest. This is obviously related to the activity of the promoter driving the expression of the introduced cDNA. If it is a constitutive promoter, its activity is low. Try to switch to other, stronger promoters. If the driving promoter is inducible, try to optimize the concentration of the appropriate inducer.

6. Some of drug selected stably transfected cell clones show 100% death during subsequent cloning or culture in the selection medium. If these clones are not frozen, it is certain that these isolated clones are false stably transfected cells. It is likely that during trypsinization and resuspension, the cells were not well suspended; instead, they aggregated. After trans-fection, some of the cells within the aggregated colonies receive fewer drugs and still undergo proliferation, forming false colonies that are picked up in the process. During the trypsinizing and picking up, these cells become loosened by physical force. Because they are not particularly stable colonies, they are killed by drug selection medium during the subsequent expanding or culture.

7. Very few frozen, stably transfected cells survive during subsequent culture. Certainly, this is due to inappropriate freezing. Make sure to use 5 to 10% (v/v) DMSO in the freezing medium.

0 0

Post a comment