( through it. RECOMBINANT LU...)Luciferase Reporter Gene Cells and Recombinant Luciferase Dilutions Studied with the CLIPR Luciferase Assay Kit and the LMax Microplate Luminometer (MaxLine Application Note #38)

RECOMBINANT LUCIFERASE ASSAY PROCEDURE
1. Diluted luciferase standards and buffer blank (10 L each) were pipetted into microplate wells in quadruplicate. The final luciferase content ranged from 100 fg/well to 100 ng/well for the dynamic range standard curve and 6.25 to 200 fg/well for the low-end standard curve.

2. For the fast kinetic assays, the Lmax was set to inject 100 uL (injector M) and then to immediately begin integrating at consecutive 1-sec intervals.

3. For the endpoint assays, the Lmax was set up to inject 100 L/well, followed by a 2-second delay and a 1-second read.

PREPARATION OF CELLS AND ASSAY OF CELLULAR LUCIFERASE
1. PCRE-luc-CHOK1 cells were split into 96-well clear-bottom white microplates at 20,000 to 100,000 cells/well (100 L/well). Prior to use, they were treated with or without 1 mM cAMP for 4 hrs.

2. For the fast kinetic assays, the Lmax was set to inject 100 uL (injector M) and then to immediately begin integrating at consecutive 1-sec intervals.

3. For the endpoint assays, the Lmax was set up to inject 100 L/well, followed by a 2-second delay and a 1-second read.

REACTIONS PROFILES
The response of recombinant firefly luciferase to the addition of reagent is shown in Figure 2. The flash of light reached its maximum within 2 seconds and decayed thereafter.

The response of PCRE-luc-CHOK1 cells to the addition of reagent is shown in Figure 3. cAMP-treated cells (upper plot) had 5-fold increase in signal compared to the untreated controls (lower plot). The kinetic profiles were remarkably similar to that of recombinant luciferase: the light intensity was maximal within approximately 2 seconds and decayed thereafter to a much lower steady state. These results indicated that the CLIPR Luciferase Assay caused esse
'"/>

Source: