Luciferase assay

 Dual- Luciferase Gene Reporter Assay
 

A - Cell Lysis

1. Prepare the Passive Lysis Buffer (PLB) just before use. Prepare sufficient quantity of 1XPLB by adding 1 volume of 5X PLB to 4 volumes of Milli-Q Water.
   For a 24-well plate dispense 100ul of 1XPLB per well.
2. Remove growth medium from cultured cells.
3. Gently apply 500ul of PBS.
4. Swirl plate and remove solution before adding PLB.
5. Shake plate at RT for 20 minutes at 200rpm. (Optional: at this point you can freeze the lysate at -20°C)
    

B - Dual-Luciferase Reporter Assay Protocol

   1. Prepare the following solutions in 15 ml falcons:

*Calculated for a 24MW plate

**Add 500 µl in excess to prime the tubes

   2. Equilibrate Solution A and B at RT for 30min in the dark. 

   3. Transfer 30ul of cell lysate to one well of a 96 well white plate. (If the lysate was frozen prior to measurement, make sure the lysate is also equilibrated at RT)

   4. Spin down the plate for 30sec at max. speed (4000rpm).

   5. At the Promega Luminometer: switch ON luminometer and "iPad".

   6. Start Glomax Software. 

   7. Put Injector 1 into solution A and Injector 2 into solution B.

   8. Place injector tips inside their respective solution tubes, this way you do not waste any solution when priming the injectors.

   9. Settings > Tools > Injectors > Prime both injectors

   You can prime injectors a second time if solutions do not fill the tubes completely.

   Put the injector tips back. Careful not to switch the injectors.

   10. Go back to main screen and click "Protocols". 

   Chose Dual-Luciferase Reporter Assay homemade.

   11. On the upper right corner you have the option to insert your plate set up.

   12. When you are ready to start click on the door symbol on the right upper corner, place your plate and close the door by clicking on the same symbol again.

   13. START. Turn off the lights in the room while measuring.

   14. When the measurement is finished, export the results.

   15. Replace your plate for the trash tray.

   16. Place the injectors in the falcon with Milli-Q Water.

   17. Settings > Tools > Injectors > Flush > 3 cycles

   18. Place the injectors in the falcon with 70% EtOH and flush twice.

   19. Place the injectors in the Milli-Q Water again and flush once.

   20. Flush both injectors with air twice.

   21. Remove trash tray and wash it.

   22. Switch off luminometer.

    Data is stored at Local Disk C > Exports 

Dual-Luciferase Reporter Assay homemade program:

luciferase solutions

A- Firefly Buffer

 Dual- Luciferase Gene Reporter Assay

1,4-Dithiothreitol (DTT, Carl Roth #6908.1, 5g) (Stored at 4°C)

Coenzyme A (NanoLight #309-500mg) (Stored at -20°C, Stocks box)

ATP (Sigma-Aldrich #A2383-1g) (Stored at -20°C, Stocks box)

Luciferin (Regis Technologies, #1-360243-200)(Stored at -20°C, Stocks box)

To prepare 10mL of 500mM DTT:

• Dissolve 771mg of DTT in 7mL of milli-Q water
• Fill up to 10mL (around 2.5mL milli-Q water)
• Prepare 70µl ready-to-use aliquots
• Store at -80°C

To prepare 10mL of 10mM Coenzyme A:

• Dissolve 76.8mg of Coenzyme A in 6mL of milli-Q water
• Fill up to 10mL
• Prepare 138µl ready-to-use aliquots
• Store at -80°C

To prepare 500ul of 100mM ATP:

• Dissolve 27.6mg ATP in 500ul of milli-Q water
• Prepare 11µl ready-to-use aliquots
• Store at -80°C

To prepare 80mg/ml D-Luciferin (Sodium Salts):

• To 100mg of D-Luciferin add 1.25mL of milli-Q water
• Dissolve on ice with dim lights
• Prepare 12µl ready-to-use aliquots
• Store at -80°C

To prepare 500mL of Lysis Buffer B:

• 8.52g Tris
• 1.27g Tris Hydrochloride
• 4mL of 3.75M NaCl
• 1.5mL 1M MgCl2
• Fill up to 500mL with milli-Q Water
• Prepare 30x 15mL aliquots
• Store at RT

B- Renilla Buffer

Ataluren (Selleck Chemicals, purchased by Biozol #SEL-S6003-10mg)

Coelenterazine h (Nano Light #301-1mg) (Stored at -80°C, Luciferase Stocks box)

To prepare 10mM PTC124:

• Solve 10mg of Ataluren in 3.518 mL DMSO
• Prepare 20µl ready-to-use aliquots
• Store at -80°C

To prepare 2mM h-CTZ:

• Protect from light
• Solve 1mg of Coelenterazine h in 1.226mL of absolute ethanol (or 500ug in 613ul absolute ethanol, CHECK THE TUBE!)
• Prepare 17µl ready-to-use aliquots
• Store at -80°C
   

To prepare 500mL of Renilla Salts:

• Solve 6.69g of Sodium Pyrophosphate x10H2O in 300mL of milli-Q water
• Add 41.64g of NaCl and let it solve
• Add 45mL of 0.5M Na₂EDTA
• Fill up to 500mL with milli-Q water and let it solving until it becomes clear
• Prepare 30x 15mL ready-to-use aliquots
• Store at RT

NOTES:

- All reagents are in the -20°C left fridge > Stocks drawer > box “Luciferase Reagents”

- DTT is in the 4°C general fridge, luciferase is written on the bottle

- I did not filter sterilize the solutions since they are only used for luciferase and most times one time use only.

- The buffers were prepared from autoclaved solutions used for Luciferase only.