Difference between revisions of "Luciferase Assay"
From Bridges Lab Protocols
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==Materials== | ==Materials== | ||
*Dual Luciferase Reporter Assay System (Promega # E1910) | *Dual Luciferase Reporter Assay System (Promega # E1910) | ||
| − | *Passive Lysis Buffer (PLB | + | *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 |
| − | *Luciferase Assay Reagent (LARII ) at -70 | + | *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 | + | *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) | *Plate Reader (Book ahead for about 30 min total) | ||
| Line 12: | Line 12: | ||
#Prepare 1X PLB using 5X stock and water | #Prepare 1X PLB using 5X stock and water | ||
#Wash wells once with 100 ul D-PBS -/- | #Wash wells once with 100 ul D-PBS -/- | ||
| − | #Add 20 uL PLB to well and incubate on a shaker for 15 min at | + | #Add 20 uL PLB to well and incubate on a shaker for 15 min at 4 degree |
| − | #Prepare Stop and Glo Reagent (dilute from 50X in Stop and Glo Buffer). Need 100 uL per | + | #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 |
#Set plate reader to luminesence | #Set plate reader to luminesence | ||
#Ensure correct measurement head is installed (one light tube) and it is set to do a top read | #Ensure correct measurement head is installed (one light tube) and it is set to do a top read | ||
#Set temperature control to off | #Set temperature control to off | ||
#Set plate layout under protocol button for both Luciferase Assay I and Luciferase Assay II | #Set plate layout under protocol button for both Luciferase Assay I and Luciferase Assay II | ||
| − | #Add 100 uL of LARII | + | #Add 100 uL of LARII 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 | #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 | #Calculate relative luciferase activity by dividing results from Assay I by Assay II | ||
Revision as of 18:35, 25 November 2009
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
- Transfect cells with reporter and pRL vector (50:1) and plate in a 96 well white plate
- Treat cells as required
- 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 4 degree
- 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
- 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 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
