3-Cyano-7-ethoxycoumarin is cytochrome P450 (CYP) isoforms CYP1A1 and CYP1A2 .

Instructions
1. Solution preparation:
3-Cyano-7-ethoxycoumarin is usually provided in powder form. It can be dissolved in an appropriate solvent (such as DMSO) to prepare a 1-10 mM stock solution.
2. Experimental procedures:
1) Preparation of enzyme reaction system: Add 3-Cyano-7-ethoxycoumarin to the reaction system containing cytochrome P-450 enzyme. The common working concentration is 10-100 µM, but the concentration can be optimized according to the experimental requirements.
2) Appropriate cofactors (such as NADPH) are usually required in the reaction system because cytochrome P-450 requires NADPH as a reducing agent.
3. Enzyme reaction: The reaction system is reacted at an appropriate temperature (usually 37°C) for a period of time, usually 30-60 minutes, and the specific time can be adjusted according to the experimental design.
4. Product detection: After the reaction is completed, the blue fluorescence of the product is detected by a fluorescence spectrophotometer or fluorescence microscope. The excitation wavelength is 410 nm and the emission wavelength is 485 nm, which produces blue fluorescence.
5. Data Analysis:
Based on the intensity of the fluorescence signal, the activity of the P-450 enzyme can be analyzed. Higher fluorescence intensity is usually associated with higher enzyme activity.

The above information is based on published literature. Experimental procedures should be appropriately modified to meet specific research demands.

DMSO: 16.5 mg/mL (76.67 mM), Sonication is recommended.