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Difference between revisions of "Luciferase Assay"

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==Materials==
 
==Materials==
−
*Dual Luciferase Reporter Assay System (Promega # E1910)
+
*Dual Glo Reporter Assay System (Promega # E2910)
−
*Passive Lysis Buffer (PLB at 5X; Promega # E1941) at -20
+
*To prepare both of these buffers resuspend the lyophylized solution and aliquot in -80
−
*Luciferase Assay Reagent (LARII ) at -70, use fresh aliquot, thaw at room temp and mix
+
*Dual-Glo Luciferase Buffer
−
*Stop and Glo Reagent and Buffer at -20
+
*Stop & Glo Buffer
−
*Plate Reader (Book ahead for about 30 min total)
+
*Dual Glo Stop & Glo Substrate (Molecular Biology Stuff at -20)
 +
*D-PBS
 +
*Cells transfected with luciferase reporter
 +
*Tube-based luminometer (GloMax 20/20)
  
  
 
==Protocol==
 
==Protocol==
−
*Transfect cells with reporter and pRL vector (50:1) and plate in a 96 well white plate
+
#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
+
#Treat cells as required
−
*Prepare 1X PLB using 5X stock and water
+
#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 100 ul D-PBS -/-
+
#Wash wells once with 1 mL D-PBS -/-, aspirate PBS.  Can freeze the cells at this point if needed.
−
*Add 20 uL PLB to well and incubate on a shaker for 15 min at RT
+
#Add 100 uL PBS and 100 uL Dual-Glo Buffer to each well
−
*Prepare Stop and Glo Reagent (dilute from 50X in Stop and Glo Buffer).  Need 100 uL per assay.  Reagent and LARII should be at room temperature
+
#Incubate on rocker for at least 10 minutes
−
*Set plate reader to luminesence
+
#Transfer liquid from each well (200 uL) into eppendorf tubes
−
*Ensure correct measurement head is installed (one light tube) and it is set to do a top read
+
#Set luminometer to measure at a 10s integration.
−
*Set temperature control to off
+
#Measure each tube individually recording the values, or saving it o excel via the GloMax software
−
*Set plate layout under protocol button for both Luciferase Assay I and Luciferase Assay II
+
#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 of LARII to lysates and read luminescence (2s delay, 10s measurement using Luciferase Assay I
+
#Add 100 uL to each tube and mix
−
*Add 100 uL of Stop and Glo Reagent and read luminescence again using Luciferase Assay II
+
#Incubate at least 10 minutes
−
*Calculate relative luciferase activity by dividing results from Assay I by Assay II
+
#Measure the Renilla luminesence in the same order
 +
#Calculate relative luciferase activity by dividing results from the Luciferase Assay over the Renilla Assay
 +
 
 +
[[ Category: Luciferase ]]
 +
[[ Category: Cell Culture ]]
 +
[[ Category: Tissue Culture ]]
 +
[[ Category: Promoters ]]
 +
[[ Category: Transcription ]]

Latest revision as of 14:30, 27 February 2017

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

  1. 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
  2. Treat cells as required
  3. Thaw out enough Dual Glo Buffer and Stop & Glo Buffer for the assay. You will need 100 uL per well of each.
  4. Wash wells once with 1 mL D-PBS -/-, aspirate PBS. Can freeze the cells at this point if needed.
  5. Add 100 uL PBS and 100 uL Dual-Glo Buffer to each well
  6. Incubate on rocker for at least 10 minutes
  7. Transfer liquid from each well (200 uL) into eppendorf tubes
  8. Set luminometer to measure at a 10s integration.
  9. Measure each tube individually recording the values, or saving it o excel via the GloMax software
  10. 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
  11. Add 100 uL to each tube and mix
  12. Incubate at least 10 minutes
  13. Measure the Renilla luminesence in the same order
  14. Calculate relative luciferase activity by dividing results from the Luciferase Assay over the Renilla Assay
Last modified on 27 February 2017, at 14:30