Cytochrome P450 Reductase Activity Assay Kit (Colorimetric) (ab204704)
Key features and details
- Assay type: Enzyme activity
- Detection method: Colorimetric
- Platform: Microplate reader
- Sample type: Cell Lysate, Microsomes, Purified protein, Tissue Lysate
- Sensitivity: 0.2 mU/well
Overview
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Product name
Cytochrome P450 Reductase Activity Assay Kit (Colorimetric)
See all Cytochrome P450 Reductase kits -
Detection method
Colorimetric -
Sample type
Cell Lysate, Microsomes, Purified protein, Tissue Lysate -
Assay type
Enzyme activity -
Sensitivity
0.2 mU/well -
Species reactivity
Reacts with: Mammals, Other species -
Product overview
Cytochrome P450 Reductase Activity Assay Kit (Colorimetric) (ab204704) couples the oxidation of NADPH by cytochrome P450 reductase (CPR) to the reduction of a nearly colorless probe into a brightly colored product with an absorbance peak at OD=460 nm, with the rate of color generation being directly proportional to CPR activity. The NADPH utilized by CPR is generated in situ from β-NADP+ via oxidation of glucose-6-phosphate (G6P) to 6-phospho-D-glucono-1,5-lactone by glucose-6-phosphatase dehydrogenase (G6PDH). The kit can be used to determine CPR activity in a variety of samples, with a detection limit of ~0.2 mU of CPR activity per reaction.
For assessment of CPR activity in crude biological samples that may have extraneous reductases capable of reducing the substrate, an inhibitor of NADPH-dependent flavoproteins is included. In this case, the specific CPR activity may be calculated by running parallel reactions in the presence and absence of inhibitor and subtracting any residual activity detected with the inhibitor present. The kit contains sufficient reagents for performing 100 reactions in a 96-well plate format.
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Notes
NADPH-cytochrome P450 reductase (CPR, EC 1.6.2.4) is a ~78 kDa membrane-bound flavoenzyme that catalyzes the transfer of electrons from NADPH to members of the cytochrome P450 monooxidase (CYP) enzyme family in the endoplasmic reticulum. CPR contains two tightly bound flavin cofactors, FAD and FMN, which participate in the sequential transfer of electrons from NADPH→FAD→FMN→CYP, oxidizing NADPH to NADP+ and reducing the CYP heme moiety to the substrate- and oxygen-binding ferrous state. As CPR is required for the function of all CYP isozymes, it plays a critical role in the metabolism of drugs, organic pollutants and other xenobiotic compounds, in addition to its role in biosynthesis of certain vitamins and steroid hormones.
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Platform
Microplate reader
Properties
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Storage instructions
Store at -20°C. Please refer to protocols. -
Components 100 tests CPR Assay Buffer 1 x 50ml G6P Standard 1 vial G6P Standard Developer 1 vial Human CPR Positive Control 1 vial Inhibitor (Diphenyleneiodonium Chloride, 10 mM) 1 x 100µl NADPH Substrate Mix 1 vial -
Research areas
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Function
This enzyme is required for electron transfer from NADP to cytochrome P450 in microsomes. It can also provide electron transfer to heme oxygenase and cytochrome B5. -
Involvement in disease
Defects in POR are the cause of adrenal hyperplasia variant type (AHV) [MIM:201750]; also known as Antley-Bixler syndrome-like phenotype with disordered steroidogenesis. AHV is a rare variant of congenital adrenal hyperplasia. It is an autosomal recessive disorder with apparent combined P450C17 and P450C21 deficiency. Affected girls are born with ambiguous genitalia, but their circulating androgens are low and virilization does not progress. Conversely, affected boys are sometimes born undermasculinized. Boys and girls can also present with bone malformations, in some cases resembling the pattern seen in patients with Antley-Bixler syndrome.
Defects in POR are a cause of isolated disordered steroidogenesis (IDS) [MIM:201750]. -
Sequence similarities
In the C-terminal section; belongs to the flavoprotein pyridine nucleotide cytochrome reductase family.
Contains 1 FAD-binding FR-type domain.
Contains 1 flavodoxin-like domain. -
Cellular localization
Endoplasmic reticulum membrane. Anchored to the ER membrane by its N-terminal hydrophobic region. - Information by UniProt
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Alternative names
- CPR
- CYPOR
- Cytochrome p450 oxidoreductase
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Images
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Cytochrome P450 Reductase Activity Assay Kit (Colorimetric) (ab204704)
Typical G6P Standard curve using Cytochrome P450 Reductase Activity Assay Kit (Colorimetric) (ab204704). One mole G6P corresponds to one mole of β-NADP+ reduced to NADPH, which subsequently generates one mole of reduced substrate.
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Cytochrome P450 Reductase Activity Assay Kit (Colorimetric) (ab204704)Reaction kinetics of recombinant human CPR (positive control) and rat microsomal CPR (with and without inhibitor).
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Cytochrome P450 Reductase Activity Assay Kit (Colorimetric) (ab204704)Relative CPR activity detected in rat liver microsomes (RLM, 25 µg total protein) and HepG2 cell lysate (40 µg total protein).