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Dual-Luciferase Quantitation in the LMax Microplate Luminometer (MaxLine Application Note #39)

PREPARATION OF REAGENTS
1. Luciferase assay reagent II (LAR II) was prepared by resuspending the lyophilized luciferase assay substrate (E151A) in 10 mL luciferase assay buffer II (E1955).

2. The Stop & Glo reagent was prepared by first reconstituting the dried Stop & Glo substrate with 200 L Stop & Glo Substrate Solvent (E463A). After gently vortexing for 10 sec, the entire reconstituted substrate was mixed with 10 mL Stop & Glo buffer (E197A).


PREPARATION OF THE LMAX
1. The solvent delivery systems of the Lmax were both cleaned by pumping 75% ethanol through them and allowing it to stand for one hour, followed by rinsing generously with deionized water (5 washes, 99 cycles each).

2. The P injector was filled with the LAR II reagent by pumping 2 mL through it. The M injector was similarly primed with Stop & Glo Reagent.


ASSAY PROCEDURE
The kit instructions were followed, except that the measurements were made on diluted recombinant enzymes instead of lysed cells.

1. In SOFTmax PRO, Dual Read mode was selected. Both the P and M injectors were set to deliver 100 L/well, followed by a 2-second delay and a 5-second read.

2. The diluted firefly+Renilla luciferase standards and buffer blank were pipetted (20 L each) into microplate wells in quadruplicate. The final enzyme content for each ranged from 2.0 to 2 x 106 fg/well.

3. The plate containing the luciferase standards was placed into the drawer of the Lmax and the injection/read cycles initiated.

During the assay, the P injector supplies the LAR II reagent to each well a minimum of 1.6 sec ahead of the time the read head moves into position over that well. The M injector supplies Stop & Glo'"/>

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