About
Redox Protein Carbonyl Assay Kits
Protein carbonyls are biomarkers of protein oxidation that are generated by several different mechanisms during oxidative stress. They can be formed on arginine, proline, threonine, and lysine residues as a result of metal-catalysed oxidation. Protein carbonyls can also arise from the direct reaction of proteins with reactive oxygen species (ROS) or can be introduced into proteins by covalent attachment of carbonyl-containing molecules such as reducing sugars or lipid peroxidation products.
An extensive overview by Weber and colleagues (Redox Biology 5 (2015) 367–380) of different methods concluded that “ELISA techniques represent the best available method to quantify protein carbonyl concentration” and “the inclusion of external standards allows standardisation, better comparison, as well as high-throughput.”
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Protein Carbonyls
An ELISA assay, using an anti-dinitrophenylhydrazine antibody, was developed by Hendrikje Buss, Christine Winterbourn and coworkers at the Mātai Hāora - Centre for Redox Biology and Medicine, University of Otago, Christchurch, New Zealand.
The assay was recently modified by Nick Magon, to use biotin hydrazide and be antibody-free, resulting in less background and a shorter assay time.
Learn more about Mātai Hāora.
Redox Innovation is a business unit of Otago Innovation.
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Background
Redox Innovation Supported by Otago Innovation.
Using the Kit
Up to 89 samples can be analysed without replicates, although duplicate or triplicate analysis is recommended for best results. Carbonyl concentrations are calibrated against a protein that has been oxidised and standardised colourimetrically.
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Stability
Redox Innovation Protein Carbonyl Assay Kits do not need to be kept under cool conditions for short-term transportation. The kits are stable at room temperature for up to six weeks. When the kit is separated into freezer, fridge and bench components as per instructions on the leaflet, it is stable until at least the expiry date.
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Additional Information
Please check out the Kit Performance Information for information on the performance of the kit that our technical support group has provided.
PDF files of the Protein Carbonyl Assay Kit Instruction Manual and Material Safety Data Sheet (MSDS) are available for download.
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Assay Principle
Key:
Protein
Blocking agent
TMB
Carbonyl group
Streptavidin-linked horseradish peroxidase
Oxidised TMB (basic)
Oxidised TMB (acidic)
Biotin hydrazide
1.
Protein samples that have been derivatised with biotin hydrazide are added and directly bound to the ELISA plate, saturating all protein-binding sites.
2.
Free biotin hydrazide, excess protein and non-protein constituents are easily washed away, meaning minimal interference. Unoccupied binding sites are subsequently blocked.
3.
Streptavidin-linked horseradish peroxidase is bound to the biotin hydrazide-protein complex.
5.
Absorbance values are related to a standard curve prepared from serum albumin containing increasing proportions of HOCI-oxidised protein.
4.
Chromogen, containing tetramethylbenzidine (TMB) and hydrogen peroxide, is added and the horseradish peroxidase catalyses the oxidation of TMB by hydrogen peroxide to a blue product. The reaction is stopped by the addition of acid, which enhances the sensitivity and gives a yellow product that can be measured at 450 nm.