Reporter Gene Assay : CAT assay
Cell Culture and Transfection
1. Distribute 1 ml of cells into each well of 6-well culture plate
2. Incubate the cells for 24 h in CO2 incubator
3. Prepare plasmid DNA (10 g of target CAT plasmid + 4 g of pGL3-LUC control
vector/ 0.5 ml M199) and transfection reagent (pFx-7, 2 mg/ml, InVitrogen, 98 g /
0.5 ml M199) [ratio of plasmid DNA and pFx-7, 1:7 (w/w)] in separate tubes
4. Mix and incubate for 30 min at 37 C  “Transfection solution”
5. Wash the cells with M199 three times
6. Add 1 ml of transfection solution prepared above to each well
7. Incubation for 1.5 h at 37 C  “Transfection”
8. Remove transfection solution from each well, add 2 ml of complete HUVEC media
into each well and incubate the cells for 24 h in CO2 incubator  “Recovery of cells”
9. The cells are washed with HANKS and experimental HUVEC media with chemical
treatments are replenished
10. Incubation for additional 16 h in CO2 incubator
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Preparation of cell extracts and enzyme assay
1. Add 4 volumes of water to 1 volume of 5X Reporter Lysis Buffer (Promega #E3971)
to produce a 1X Reporter Lysis Buffer.
2. Remove the media from the cells to be assayed. Wash the cells twice with 1X PBS.
3. Add a sufficient volume of 1X Reporter Lysis Buffer to cover the cells (100 l for
each well of a 6-well plate). Rock the dish slowly several times to ensure complete
coverage of the cells.
4. Incubate the dish at room temperature for 15 min, slowly rocking the dish several
times halfway through the incubation period.
5. Scrape all areas of the plate surface, then tilt the dish and scrape the cell lysate to the
lower edge of the plate. Transfer the cell lysate to a microcentrifuge tube with a
pipette and place the samples on ice for 5 min.
6. Vortex the tubes for 15 sec
7. Take 15 l of cell lysates to measure Luciferase activity and heat the rest of them for
CAT assay
* Heat only those lysates for use in the CAT assay at 60 C for 10 min to inactivate
endogenous deacetylase activity [If simultaneously performing reporter assays for
other co-transfected vectors, split the lysates before heating, and heat only the
lysates intended for use in the CAT assay. This heating step does not inactivate CAT,
but may inactivate other reporter enzymes such as –galactosidase or luciferase].
8. Centrifuge all samples at 12,000 x g for 5 min at 4 C.
9. Transfer the supernatant (cell extract) to a fresh tube.
10. The extracts may be assayed directly or stored at –70C.
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Luciferase assay
1. Mix 10 l of room temperature cell extract with 100 l of room temperature
Luciferase Assay Reagent and place the reaction in a luminometer.
2. Measure the light produced for a period of 10 sec.
CAT (Chloramphenicol acetyltransferase) Assay
1. Prepare the following reaction mixture in a microcentrifuge tube.
1X Reporter Lysis Buffer
50 l
Cell extracts
50 l
1 M Tris-HCl (pH 7.8)
20 l
5 mM Acetyl CoA
20 l
[14C]-Chloramphenicol (25 Ci/ml)
3 l
2. Incubate the reactions with shaking for 4 h at 37 C.
[ or use the Thermomixer, 300 rpm, 37 C, 4 h ]
3. Vortex the samples with 1 ml of ethyl acetate for 1 min.
[or use the Thermomixer, 1,400 rpm, 25 C, 10 min ]
4. Centrifuge the tubes at 12,000 rpm for 5 min.
5. Transfer the upper, organic phase to a fresh tube and evaporate to dryness completely
using the Speed-Vac concentrator (at high temperature for about 20 min).
6. Resuspend the residue in 30 l of chloroform and spot each sample onto a silica gel
TLC plate.
7. Run the TLC plate in a developing chamber pre-equilibrated for 1 h with chloroform
–methanol (95:5, v/v)
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8. Remove the TLC plate from the chamber and dry at room temperature.
9. Autoradiography and counting radioactivities
* Chloramphenicol becomes acetylated at two sites and these two forms migrate faster
than the unmodified chloramphenicol substrate on the TLC plates in this solvent system.
After autoradiography, unmodified substrate and acetylated products can be cut from the
plate and counted in a scintillation counter for quantitation of CAT activity.
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