Resorufin benzyl ether

Guide (The following is the experimental plan we recommend. This plan serves only as a reference guide. The specific operations should be adjusted according to your actual needs.)
Measurement of CYP3A4 activity^[2]: Prepare the reaction solution.
a. The standard solution contains 1 mM Resorufin benzyl ether, which is used as the fluorescent substrate. Dissolve 5 mg of Resorufin benzyl ether in a mixture of 1 mL of 2% w/v Poloxamer 188 (HY-D1005A), 500 mL Dimethyl sulfoxide (HY-Y0320), and 3.5 mL acetonitrile.
b. Freshly prepared CYP3A4 enzyme solution. Mix 5 mL of 1 mM enzyme stock solution with 995 mL of buffer to obtain a 5 nM enzyme solution.
2. Measurement of CYP3A4 activity a. Perform the reaction in a 96-well plate. Add 99 mL of buffer mixture and 1 mL of 1 mM Resorufin benzyl ether to each well. Adjust the final concentration to 5 mM.
b. Before incubation at 37℃ for 30 minutes, transfer 100 mL of 5 nM enzyme solution.
c. Enzyme activity was determined under fluorescence detection at excitation wavelength (lex = 570 nm) and emission wavelength (lem = 590 nm).
d. Factors affecting the determination of CYP3A4 activity. Such as buffer solutions (phosphate, Tris-HCl buffer) and buffer concentrations (50-200 mM), as well as incubation time (0-50 min).

Metabolic Activity Assay of CYP3A4^[3] : Add the CYP3A4 enzyme to a final concentration of 5 pmol per well.
2. For each reaction, 50 pM of substrate and 200 mM phosphate buffer solution were used. < br / > 3. Before measuring the fluorescence of metabolites, incubate them with BzRes for 45 minutes. The excitation wavelength (Ex) is 530 nm and the emission wavelength (Em) is 590 nm.
4. The concentration of the full-strength extract is specified as 100% (diluted 1:4 in the final measurement quantity).
5. Dilute the 100% extract in a 1:3 ratio twice. Calculate the mean and standard deviation of the fluorescence values.

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.