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==Materials==
*Dual Luciferase Reporter Assay System (Promega # E1910)
*Prepare required amount of 1X Passive Lysis Buffer (PLB at 5X; Promega # E1941) 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, use fresh aliquot, thaw at room temp and mix.*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)
#Prepare 1X PLB using 5X stock and water
#Wash wells once with 100 ul D-PBS -/-
#Add 20 uL PLB to well and incubate on a shaker for 15 min at RT4 degree#Prepare Stop and Glo Reagent (dilute from 50X in Stop and Glo Buffer). Need 100 uL per assaywell in 96-well plate. Reagent and LARII should be at room temperature
#Set plate reader to luminesence
#Ensure correct measurement head is installed (one light tube) and it is set to do a top read
#Set temperature control to off
#Set plate layout under protocol button for both Luciferase Assay I and Luciferase Assay II
#Add 100 uL of LARII to plate and then add 20uL of lysates and read luminescence (2s delay, 10s measurement using Luciferase Assay I
#Add 100 uL of Stop and Glo Reagent and read luminescence again using Luciferase Assay II
#Calculate relative luciferase activity by dividing results from Assay I by Assay II
