Luciferase Assay
From Bridges Lab Protocols
Materials
- Dual Glo Reporter Assay System (Promega # E2910)
- To prepare both of these buffers resuspend the lyophylized solution and aliquot in -80
- Dual-Glo Luciferase Buffer
- Stop & Glo Buffer
- Dual Glo Stop & Glo Substrate (Molecular Biology Stuff at -20)
- D-PBS
- Cells transfected with luciferase reporter
- Tube-based luminometer (GloMax 20/20)
Protocol
- Transfect cells with pGL3/4 plasmid and pRL vector (50:1) and plate in a 12 well plate. Can also do these assays in a 48 or 96 well plate, but the volumes here are for a 12-well dish
- Treat cells as required
- Thaw out enough Dual Glo Buffer and Stop & Glo Buffer for the assay. You will need 100 uL per well of each.
- Wash wells once with 1 mL D-PBS -/-, aspirate PBS. Can freeze the cells at this point if needed.
- Add 100 uL PBS and 100 uL Dual-Glo Buffer to each well
- Incubate on rocker for at least 10 minutes
- Transfer liquid from each well (200 uL) into eppendorf tubes
- Set luminometer to measure at a 10s integration.
- Measure each tube individually recording the values, or saving it o excel via the GloMax software
- Prepare Stop & Glo reagent by adding the Dual Glo Stop & Glo Substrate to the Stop & Glo Buffer at a 1:100 dilution and mix well
- Add 100 uL to each tube and mix
- Incubate at least 10 minutes
- Measure the Renilla luminesence in the same order
- Calculate relative luciferase activity by dividing results from the Luciferase Assay over the Renilla Assay
