Overview

  • 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: Other species, Mammals
  • 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.

  • 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.

  • Platform

    Microplate reader

Properties

  • 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

  • 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
  • Alternative names

    • CPR
    • CYPOR
    • Cytochrome p450 oxidoreductase
    • DKFZp686G04235
    • FLJ26468
    • NADPH Cytochrome P450 Reductase
    • NADPH dependent cytochrome P450 reductase
    • NADPH--cytochrome P450 reductase
    • NCPR_HUMAN
    • P450 (cytochrome) oxidoreductase
    • P450 Cytochrome Oxidoreductase
    • P450R
    • por
    see all

Images

  • 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.

  • Reaction kinetics of recombinant human CPR (positive control) and rat microsomal CPR (with and without inhibitor).

  • Relative CPR activity detected in rat liver microsomes (RLM, 25 µg total protein) and HepG2 cell lysate (40 µg total protein).

Protocols

References

ab204704 has not yet been referenced specifically in any publications.

Customer reviews and Q&As

Abreviews
1. Standard curve measurement
- Set up Standard wells = 90㎕ standard dilutions.
- To each of the standard wells, add 5㎕ of NADPH Substrate Mix and 5㎕ of G6P Standard Developer to make the final bolume 100㎕ /well. Mix well.
- Incubate standard wells for at least 30 minutes at room temperature, protected from light.
- Measure absorbance in a colorimetric microplate reader at OD = 460nm at 20℃ for all of G6P standard wells.
2. Set up Reaction wells.

Component
Human
CPR (㎕)
Human CPR + Inhibitor (㎕)
Positive Inhibitor (㎕)
Background Control (㎕)
(Inhibitor)
0
x
10
2
Human CPR
5
5
5
5
CPR Assay buffer
55
up to 60
45
up to 70


3. Prepare 30㎕ of Reaction Mix for each reaction.

Component
Reaction Mix (㎕)
NADPH Substrate
5
CPR assay buffer
25


4. Add 30㎕ of Reaction Mix into each well. Mix well.
5. Incubate for 5 minutes at room temperature to allow the inhibitor to bind targets
6. Make a 20mM G6P Reaction Solution by diluting the 100mM G6P stock solution with CPR Assay Buffer at a 1:5 ratio.
7. Add 10㎕ of the 20mM G6P solution to each well except background control.
Mix well.
8. Immediately measure absorbance at OD 460nm in kinetic mode for 25-30 minutes at 25℃

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Verified customer

Submitted Oct 31 2019

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