( Evelyn McGown Ph.D. Michael Su...)Luciferase Reporter Gene Cells and Recombinant Luciferase Dilutions Studied with the CLIPR Luciferase Assay Kit and the LMax Microplate Luminometer (MaxLine Application Note #38)

Evelyn McGown, Ph.D. , Michael Su, M.S. and Jin Liao, M.D., Ph.D.
Molecular Devices Corporation, 8/00

INTRODUCTION
Luciferase from the North American firefly (Photinus pyralis) is very popular foruse in Reporter Gene Assays. The enzyme catalyzes the ATP-dependent oxidationof luciferin with the concomitant release of light (Figure 1)¹. When luciferase is the limiting component in the reaction, the amount of light emitted isproportional to the concentration of luciferase. The reaction is extremely efficientand the quantum yield is the highest of any characterized bioluminescentreaction. The bright signal makes this a valuable enzyme to use for reportingpromoter activity. The intensity of the emitted light can be measured easily withan Lmax microplate luminometer.

The CLIPRLuciferase Assay reagent was originally developed for use in aglow kinetics mode in the Molecular Devices CLIPR high throughputmicroplate luminometer. However, the reagent can also be used in a flash modeto measure cell-based luciferase in Molecular Devices Lmax microplate luminometer. Cell lysis and enzyme detection occur upon addition of the single reagent: no pre-lysis or other pre-treatment step is necessary. Results are presented below.

MATERIALS
1. Lmax microplate luminometer with SOFTmax PRO for Lmax (Molecular Devices Corp.)

2. Recombinant Firefly Luciferase, Sigma Cat # 9506.

3. CLIPR Luciferase Assay Kit, Cat. No. R 8032; Molecular Devices Corporation; 1-800-635-5577. The kit contains luciferin (lyophilized) and luciferase assay buffer.

4. PBS 1X - Dulbeccos Phosphate Buffered Saline (PBS). Irvine Scientific, Cat. #9235. Phone: 800-437-5706.

5. Solid white 96-well microplates; e.g. CorningCostar Cat. No. 3912, Tel: 1 8004921110.

6. Clear-bottomed white 96-well microplates. Cornin
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