Cytochrome P450 Reductase (CPR) Activity Assay Kit

K148

96T (80 samples)

9.11 U/L

9.11–550 U/L

This assay kit is designed to detect the enzymatic activity of cytochrome P450 reductase (CPR) in tissue and cell samples.

Store in the dark at-20 °C for 6 months.

Cytochrome P450 reductase (CPR) is a key component of the cytochrome P450 (CYP) system. After acquiring electrons from an electron donor, CPR transfers these electrons to CYPs, enabling CYPs to react with oxidized substrates and molecules to produce reduced substrates and water.

This assay kit leverages the electron-transfer capability of CPR to catalyze the conversion of substrates into a chromogenic compound, which exhibits maximum absorbance at 450 nm. By measuring the optical density (OD) value of this chromogenic compound at 450 nm within a defined detection time interval, the enzymatic activity of CPR can be quantified.

When using this kit to analyze tissue or cell samples, it is necessary to determine the total protein concentration; for animal tissue and cell samples, the BCA assay (Catalog No.: K001) is recommended; for plant tissue samples, the Coomassie Brilliant Blue assay is recommended (Catalog No.: K002).

Microplate reader (430–470 nm; optimal detection wavelength: 450 nm), 37 °C incubator, double-distilled water, and 0.9% saline solution

1.Allow the reagents to equilibrate to room temperature.

2.Preparation of Reagent 3 working solution: Take one bottle of Reagent 3 and dissolve it in 6 mL of Reagent 2, then mix thoroughly; Unused solution can be stored at 2–8 °C, protected from light, for up to 3 days.

3.Preparation of the 1 mmol/L standard solution: Take one vial of reagent 4, dissolve it in 5 mL of double-distilled water, and mix thoroughly to obtain a 1 mmol/L standard solution; Unused reagent can be stored at –20 °C for up to 7 days.

4.Preparation of the 500 μmol/L standard solution: prepare the solution at a volume ratio of 1 mmol/L standard solution: double-distilled water = 1:1; Unused reagent can be stored at −20 °C for up to 3 days.

5. Dilution of standard solutions at different concentrations:

1.Sample Processing

Cell sample preparation: Collect 1×10^6 cells, add 200 μL of physiological saline (0.9% NaCl) for homogenization, then centrifuge at 4 °C and 10,000 × g for 10 min. Collect the supernatant, place it on ice for subsequent analysis; samples analyzed on the same day are considered valid. Reserve a portion of the supernatant for protein concentration determination.

Tissue samples: Homogenize the tissue samples at a ratio of mass (g): volume (mL) of physiological saline (0.9% NaCl) = 1:9; centrifuge at 4 °C for 10 min at 10,000 × g; collect the supernatant, place it on ice for subsequent analysis, and ensure that samples analyzed on the same day are considered valid. Reserve a portion of the supernatant for protein concentration determination.

2.Sample dilution

Prior to formal testing, select 2–3 samples expected to exhibit significant differences and dilute them into varying concentrations for a preliminary experiment. Based on the results of this preliminary experiment and in accordance with the linear range of this assay kit (9.11–550 U/L), follow the dilution guidelines provided in the table below (for reference only):

Note: The diluent is normal saline (0.9% NaCl).

Standard curve: y=ax+b

Calculation formula for cytochrome P450 reductase (CPR) enzymatic activity in tissue and cellular samples:

Definition of enzyme activity: At 37 °C, one unit of enzyme activity is defined as the amount of protein per gram of tissue or cell that consumes 1 μmol of NADPH per minute in a reaction system.

CPR activity (U/gprot) = (ΔA-b) ÷ a ÷ Cpr × f ÷ T

pour:

y: OD value of the standard – blank OD value (OD value when the standard concentration is 0)

x: concentration of the standard

a: Standard curve slope

b: Intercept of the standard curve

ΔA: OD value of test wells – OD value of sample control wells

Cpr: Protein concentration of the test sample after homogenization (gprot/L)

f: Dilution factor of the sample before adding it to the detection system

T: Incubation time, 10 min

1.This assay kit is for research use only. Should it be used for clinical diagnosis or any other purpose, our company shall not be held liable for any resulting issues, nor shall it assume any legal liability.

2.Before conducting the experiment, please carefully read the instruction manual, properly calibrate the instrument, and strictly follow the instructions when performing the experiment.

3.During the experiment, please wear a lab coat and latex gloves to ensure proper personal protection.

4.The detection range of the assay kit is not equivalent to the concentration range of the analyte in the sample. If the concentration of the analyte in the sample is too high or too low, please appropriately dilute or concentrate the sample.

5.If the sample being tested is not among the sample types listed in the product instructions, it is recommended to first conduct a preliminary experiment to verify the assay's validity.

6.The final experimental results are closely dependent on factors such as the efficacy of the reagents, the operator's proper procedures, and the experimental environment. Our company assumes liability solely for the assay kits themselves and is not liable for sample consumption resulting from the use of these kits. Before use, please carefully consider the potential sample volume required and reserve sufficient sample quantities.