Luciferase reporter assay

Luciferase Reporter Assay

1. Plate cells of interest to 60-70% confluence in 3 wells of a 6 well-plate (biological triplicate)

2. Transfect cells of interest with 2µg of pGL3b-8XGTIIC firefly luciferase reporter plasmid and 200ng of the pRL-TK Renilla plasmid using Lipofectamine with Plus reagent.

The following steps utilize the Dual-Luciferase Reporter Assay from Promega

3. 48 hours after transfection, remove the growth media, wash with 1X PBS, then add 500 µL of 1X passive lysis buffer.

4. Scrape cells, collect the lysate, then incubate on the rocker at room temperature for 15 min.

5. Add 100 µL of Luciferase Assay Reagent II (LAR II) to three wells in a white-walled, clear-bottom, 96 well plate (technical triplicate). LARII should be at room temperature.

6. Transfer up to 20 µL of cell lysate into a well containing 100 µL LARII and mix by pipetting 2-3 times.

7. Record the firefly luciferase activity measurement (program the luminometer to perform a 2-second premeasurement delay, followed by a 10-second measurement period for each reporter assay)

8. Add 100 µL of Stop & Glo Reagent.

9. Record the Renilla luciferase activity

10. Normalize the firefly luciferase activity to the Renilla luciferase activity

Category