We now are using the Dual-Glo system, but for reference this was the protocol using the previous protocol with the plate reader.

Materials

• Dual Luciferase Reporter Assay System (Promega # E1910)
• Prepare required amount of 1X Passive Lysis Buffer (PLB) by adding 1 volume 5X PLB to 4 volumes of distilled water. Mix well and store at -20
• Prepare Luciferase Assay Reagent (LARII) by resuspending the lyophilized Luciferase Assay Substrate in Luciferase Assay Buffer II (10ml). Aliquot remaining and store at -70.
• Stop and Glo Reagent and Buffer at -20. Prepare required amount of Stop&Glo Reagent from 50X Stop&Glo Substrate. Add 50X Stop&Glo Substrate to final 1X concentration (i.e. 0.2ml of 50X Stop&Glo Substrate to 10ml of Stop&Glo Buffer to make a 1X solution of Stop&Glo Reagent).
• Plate Reader (Book ahead for about 30 min total)

Protocol

1. Transfect cells with reporter and pRL vector (50:1) and plate in a 96 well white plate
2. Treat cells as required
3. Prepare 1X PLB using 5X stock and water
4. Wash wells once with 100 ul D-PBS -/-
5. Add 20 uL PLB to well and incubate on a shaker for 15 min at 4 degree
6. Prepare Stop and Glo Reagent (dilute from 50X in Stop and Glo Buffer). Need 100 uL per well in 96-well plate. Reagent and LARII should be at room temperature
7. Set plate reader to luminesence
8. Ensure correct measurement head is installed (one light tube) and it is set to do a top read
9. Set temperature control to off
10. Set plate layout under protocol button for both Luciferase Assay I and Luciferase Assay II
11. Add 100 uL of LARII plate and then add 20uL of lysates and read luminescence (2s delay, 10s measurement using Luciferase Assay I
12. Add 100 uL of Stop and Glo Reagent and read luminescence again using Luciferase Assay II
13. Calculate relative luciferase activity by dividing results from Assay I by Assay II