Luciferase Reporter Assay – Protocol

LUCIFERASE ASSAY

Harvesting the cell (adherent cells)

1. Remove the culture medium
2. 1ml of PBS is added to each well.
3. Scrape out the cells and transfer it to Eppendorf tube.

Floating cells

1. Take 1 ml of culture medium (which has cell) in an Eppendorf tube.

Cell lysis

1. A short centrifuge (18-20 seconds) is done.
2. The pellets are obtained in the bottom of the tube.
3. Carefully vacuum out the liquid leaving the pellet behind.
4. 100 ml of CCLR lysis buffer is added to the pellet
5. Vortex to mix. Keep the tube in the ice to keep it cold in between successive vortex.
6. After the pellets dissolve; incubate in ice for 10 minutes.
7. Spin at 12000rpm for 20 seconds in 4°C.
8. Keep the 96 well plate for luciferase (OPAQUE PLATE) on ice.
9. 10µl of supernatant is added to each well of 96 well plate.
10. Now the mixture is ready for measurement.

LUCIFERASE ACTIVITY MEASUREMENT

1. Just before the measurement add 50 µl of luciferase substrate to each well.
2. Measure the activity using spectrophotometer.