Dual Luciferase assay reagent Protocol    

Protocol for use of DLR assay reagent:

Use Targeting Systems CLR according to the protocol found here. Assay 20 µl cell lysate with 50 µl GAR reagent included in the DLR system see protocol for GAR reagents below. Read in luminometer. Next add 100 µl FLAR reagent included in the DLR system. Incubate sample at Room Temp. for 5 minutes and read sample in luminometer for Firefly luciferase activity. 

CLR Protocol:

Dilute the 5X Cell lysis reagent to 1X with water.  Aspirate supernatant cell culture media.  Add enough cell lysis reagent to cover the cells well (150 µl per well of a 24-well dish, 250 µl per well of a 12-well dish, 800 µl per well of a 6-well dish, 2.5 ml per 10 cm dish.  Keep cells of an orbital shaker for 15-20 mins .  Use 5-20 µl of the lysate for assay.

GAR Protocol:

Dilute 100X coelenterazine substrate with assay dilution

buffer  supplied with each product just before use. 

Assay 20 µl of the gaussia luciferase sample with 50 µl Gaussia luciferase assay reagent. If activity is too high dilute with serum-free DMEM but use  20 µl total volume per assay using 50 µl of Gaussia luciferase assay reagent.

Mix well and read in the luminometer.