Overview

  • Product name

    Human Complex IV ELISA Kit
    See all Complex IV kits
  • Detection method

    Colorimetric
  • Precision

    Intra-assay
    Sample n Mean SD CV%
    HeLa Extract 8 2.8%
    Inter-assay
    Sample n Mean SD CV%
    HeLa Extract 3 7.7%
  • Sample type

    Cell culture extracts, Tissue Extracts
  • Assay type

    Sandwich (quantitative)
  • Sensitivity

    64 µg/ml
  • Range

    65.84 µg/ml - 500 µg/ml
  • Recovery

    Sample specific recovery
    Sample type Average % Range
    Fetal Bovine Serum 150 123% - 178%

  • Assay time

    1h 30m
  • Assay duration

    One step assay
  • Species reactivity

    Reacts with: Human
  • Product overview

    Complex IV in vitro SimpleStep ELISA® (Enzyme-Linked Immunosorbent Assay) kit is designed for the accurate quantitative measurement of Complex IV protein in human cell and tissue extracts.


    The SimpleStep ELISA® employs an affinity tag labeled capture antibody and a reporter conjugated detector antibody which immunocapture the sample analyte in solution. This entire complex (capture antibody/analyte/detector antibody) is in turn immobilized via immunoaffinity of an anti-tag antibody coating the well. To perform the assay, samples or standards are added to the wells, followed by the antibody mix. After incubation, the wells are washed to remove unbound material. TMB substrate is added and during incubation is catalyzed by HRP, generating blue coloration. This reaction is then stopped by addition of Stop Solution completing any color change from blue to yellow. Signal is generated proportionally to the amount of bound analyte and the intensity is measured at 450 nm.Optionally,instead of the endpoint reading, development of TMB can be recorded kinetically at 600 nm.

  • Notes

    Range of complex IV / cytochrome c oxidase assay kits

    Biochemical assay - ab239711

    Immunocapture with biochemical assay (plate-based) - ab109911 (rodent)  and ab109909 (human)

    Immunocapture with biochemical assay (dipstick) - ab109878 (rodent) and ab109876 (human)

    Immunocapture with biochemical assay and ELISA - ab109910 (human) 

    ELISA - ab179880 (human) (this kit)


    Background info on Complex IV

    Complex IV, also called cytochrome c oxidoreductase or cytochrome c oxidase, is the terminal proton pump of the mitochondrial electron transport chain where it transfers electrons from reduced cytochrome c to molecular oxygen while pumping protons across the inner mitochondrial membrane. Apart from proton translocation to the intermembrane space, Complex IV also translocates protons to its catalytic site to reduced dioxygen into water.

    Complex IV is composed of 13 different subunits, three of which (I, II and III) are encoded in the mitochondrial DNA and the remainder in the nuclear DNA. Complex IV contains four prosthetic groups, two heme groups (a and a3) and two copper atoms (CuB and CuA). Heme a3 and CuB form a bimetallic center where oxygen reduction is coupled to proton movement (also known as redox linkage). CuA, on the other hand, shuttles electrons from reduced cytochrome c to Heme a before transferring to the bimetallic center.

    Functional Complex IV is not only dependant on the presence of all 13 subunits of the complex, but also on the assembly of subunits inside the inner mitochondrial membrane and insertion of prosthetic groups within the catalytic core of the enzyme. These functions are dependent on non structural proteins encoded in the nuclear DNA.

    Biochemical defects of this enzyme complex are a common cause of OXPHOS diseases and have been associated with classical syndromes such as MERRF (Myoclonic Epilepsy and Ragged Red Fibers), MELAS (Mitochondrial encephalopathy, lactic acidosis and stroke-like symptoms), Leigh Syndrome and MLASA (Mytochondrial myophathy and sideroblastic anaemia). At the molecular level, Complex IV defects have been found as a consequence of genetic mutations in genes encoding for (1) structural subunits, (2) proteins important for mitochondrial DNA transcription, RNA translation or maintenance (such as LRPPRC, EFG1mt, MRPS16, PUS1, TACO1, POLG amongst others) or (3) post-translational assembly factors (such as SURF1, SCO1, SCO2, COX10, COX15). The levels of Complex IV have also been found altered in patients with Alzheimer’s disease and during prolonged hypoxia.

  • Tested applications

    Suitable for: Sandwich ELISAmore details
  • Platform

    Microplate

Properties

Applications

Our Abpromise guarantee covers the use of ab179880 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
Sandwich ELISA Use at an assay dependent concentration.

Images

  • SimpleStep ELISA technology allows the formation of the antibody-antigen complex in one single step, reducing assay time to 90 minutes. Add samples or standards and antibody mix to wells all at once, incubate, wash, and add your final substrate. See protocol for a detailed step-by-step guide.

     

  • The HeLa lysate range was prepared as described in Section 9 of the protocol using a dilution factor of 1.4. Raw data values are shown in the table. Background-subtracted data values (mean +/- SD) are graphed.

  • Background subtracted data from duplicate measurements are plotted.

  • Background subtracted data from duplicate measurements are plotted.

  • The levels of CIV in 143B-derived Rho0 cells were measured in comparison to the levels of 143B-Wild type cells by loading in triplicate 300µg/mL of protein per well. The concentrations of CIV were interpolated from a Complex IV standard curve of HeLa cell extract and graphed in percent relative to Complex IV expression in HeLa cell extract. The concentration of Complex IV in the Rho0 cell line was not detectable.

  • The Levels of CIV deficient fibroblasts with a compound heterozygote mutation in the SURF1 gene >c.311insAT_delTCTGCC
    AGCC, c.751C>Tp.R251X (kind gift from Dr. Garry K. Brown, University of Oxford) were measured in comparison to the levels of a control primary fibroblast by loading in triplicate 300µg/mL of total protein per well. The concentrations of CIV were interpolated from a standard curve of HeLa cell extract and graphed in percent relative to Complex IV expression in the HeLa cell extract. The concentration of Complex IV in the cell line with the SURF1 mutation was 16% from the control fibroblast.

Protocols

References

This product has been referenced in:

  • Jaworski S  et al. Degradation of Mitochondria and Oxidative Stress as the Main Mechanism of Toxicity of Pristine Graphene on U87 Glioblastoma Cells and Tumors and HS-5 Cells. Int J Mol Sci 20:N/A (2019). Read more (PubMed: 30717385) »
  • Andreux PA  et al. Mitochondrial function is impaired in the skeletal muscle of pre-frail elderly. Sci Rep 8:8548 (2018). Read more (PubMed: 29867098) »
See all 3 Publications for this product

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