https://bridgeslab.sph.umich.edu/protocols/api.php?action=feedcontributions&feedformat=atom&user=CSherryBridges Lab Protocols - User contributions [en]2026-04-22T04:25:28ZUser contributionsMediaWiki 1.45.1https://bridgeslab.sph.umich.edu/protocols/index.php?title=Luciferase_Assay&diff=428Luciferase Assay2009-11-25T18:35:03Z<p>CSherry: </p>
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<div>==Materials==<br />
*Dual Luciferase Reporter Assay System (Promega # E1910)<br />
*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<br />
*Prepare Luciferase Assay Reagent (LARII) by resuspending the lyophilized Luciferase Assay Substrate in Luciferase Assay Buffer II (10ml). Aliquot remaining and store at -70.<br />
*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).<br />
*Plate Reader (Book ahead for about 30 min total)<br />
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==Protocol==<br />
#Transfect cells with reporter and pRL vector (50:1) and plate in a 96 well white plate<br />
#Treat cells as required<br />
#Prepare 1X PLB using 5X stock and water<br />
#Wash wells once with 100 ul D-PBS -/-<br />
#Add 20 uL PLB to well and incubate on a shaker for 15 min at 4 degree<br />
#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<br />
#Set plate reader to luminesence<br />
#Ensure correct measurement head is installed (one light tube) and it is set to do a top read<br />
#Set temperature control to off<br />
#Set plate layout under protocol button for both Luciferase Assay I and Luciferase Assay II<br />
#Add 100 uL of LARII plate and then add 20uL of lysates and read luminescence (2s delay, 10s measurement using Luciferase Assay I<br />
#Add 100 uL of Stop and Glo Reagent and read luminescence again using Luciferase Assay II<br />
#Calculate relative luciferase activity by dividing results from Assay I by Assay II</div>CSherry