1. Dual-Luciferase Reporter Assay System kit, Cat #E1960; Promega Corporation. The contents include: Luciferase assay buffer II (E1955); Luciferase assay substrate (E151A), Stop & Glo buffer (E197A); Stop & Glo substrate (E193B); Stop & Glo Substrate Solvent (E463A); Passive lysis buffer, 5x reagent (E194A).
2. Firefly Luciferase, Sigma Cat # 9506.
3. Recombinant Renilla luciferase; Novalite, Cat #4400; Chemicon International; 800-437-7500.
4. Solid white microplates; e.g. CorningCostar Cat. No. 3912, Tel: 1800492 1110.
5. Lmax microplate luminometer with SOFTmax PRO for Lmax (Molecular Devices Corp.)
PREPARATION OF LUCIFERASE STANDARDS
1. Passive lysis buffer was prepared by mixing 2 mL of passive lysis 5x reagent (E194A) with 8 mL deionized water.
2. The firefly luciferase stock (1 mg/mL) was prepared in phosphate-buffered saline (PBS) containing 1% bovine serum albumin (BSA) and frozen in aliquots at -70C until use. When needed, a vial was thawed and a 10 g/mL standard prepared by diluting 10 L of the stock with 990 L passive lysis buffer.
3. The Renilla luciferase stock (10 g/mL) was prepared by adding 990 L passive lysis buffer to a 10 g vial of the enzyme and mixing gently. The solution (10 g/mL) was divided into aliquots and frozen at -70C until use.
4. The dual luciferase working standard #1 was prepared by diluting 10 L each of the 10 g/mL firefly and 10 g/mL Renilla luciferase with 980 L passive lysis buffer (concentration of each enzyme = 100 ng/mL). Serial one-to-ten dilutions in passive lysis buffer achieved working standard concentrations from 105 to 10-1 pg/mL for both enzymes. The diluted standards were kept on ice until use.