Renilla glo luciferase assay buffer

Example 8

Luminescence Assay Procedure:

Each compound to be screened was diluted to a concentration of 100 uM in Renilla Glo Luciferase Assay buffer (Promega Corporation) and then further diluted to 2×serial dilution in Renilla Glo Luciferase Assay buffer. NANOLUC® luciferase and Renilla luciferase were diluted to a concentration of 4 ng/ml in OptiMEM+0.1% FBS. 50 ul of each compound dilution was then mixed with 50 ul of each enzyme dilution and incubated for 1 minute at room temperature. Light output for each compound was measured on a GLOMAX®-Multi+ luminometer. Km and Vmax were calculated for each enzyme/substrate combination using a GraphPad Prism Michaelis-Menten non-linear fit (FIGS. 1A-ID).

FIGS. 1A-ID demonstrate that Renilla luciferase produced ˜7×more light with Coelenterazine-H compared to Furimazine. NanoLuc® luciferase produced significantly more light than Renilla with both furimazine and Coelenterazine-H.

Example 8

Luminescence Assay Procedure:

Each compound to be screened was diluted to a concentration of 100 uM in Renilla Glo Luciferase Assay buffer (Promega Corporation) and then further diluted to 2× serial dilution in Renilla Glo Luciferase Assay buffer. NANOLUC luciferase and Renilla luciferase were diluted to a concentration of 4 ng/ml in OptiMEM+0.1% FBS. 50 ul of each compound dilution was then mixed with 50 ul of each enzyme dilution and incubated for 1 minute at room temperature. Light output for each compound was measured on a GLOMAX®-Multi+ luminometer. Km and Vmax were calculated for each enzyme/substrate combination using a GraphPad Prism Michaelis-Menten non-linear fit (FIGS. 1A-1D).

FIGS. 1A-1D demonstrate that Renilla luciferase produced ˜7× more light with Coelenterazine-H compared to Furimazine. NanoLuc® luciferase produced significantly more light than Renilla with both furimazine and Coelenterazine-H.