Description
Key facts
Detection method:Colorimetric,
Sample types:Serum, Plasma, Cell culture extracts, Tissue Homogenate,
Assay type:Quantitative,
Sensitivity:= 0.015 µM,
Assay time:1h 30m,
Assay Platform:Microplate reader
Product details:
Protein Carbonyl Content Assay Kit ab126287 is designed to provide a simple and accurate method of quantifying carbonyls in protein samples.
The protein carbonyl assay protocol is based on the reaction of DNPH with protein carbonyls. This is the most popular assay type for measuring protein carbonyl content. DNP hydrazones formed in this reaction are easily quantifiable at 375 nm absorbance.
When used with, for example a 1 mg (~15 nmol) sample, this protein carbonyl assay method has a detection limit of about 0.15 nmol carbonyl. For context, BSA typically contains approximately 1-3 nmol carbonyl/mg.
Protein carbonyl assay protocol summary:
- add DNPH to samples and incubate for 10 min
- add TCA to samples, incubate for 5 min, spin for 2 min, and discard supernatant
- wash pellet by sonicating in acetone, spin and remove acetone, repeat acetone wash step
- add Guanidine solution and resolubilize pellet, and transfer to plate
- analyze with a microplate reader to measure protein carbonyl content, also perform a protein assay (eg. BCA assay) on samples to establish protein concentration
The Safety Datasheet for this product has been updated for certain countries. Please check the current version in the Support and downloads section.
Properties and Storage Information:
Shipped at conditions-Blue Ice, Appropriate short-term storage conditions--20°C, Appropriate long-term storage conditions--20°C, Storage information-+4°C
Supplementary Information:
This supplementary information is collated from multiple sources and compiled automatically.
Protein carbonyls also known as carbonylated proteins serve as key markers for oxidative stress. They possess a carbonyl group introduced through oxidative modification of susceptible amino acid residues like lysine threonine proline and arginine. Protein carbonyls have no specific mass due to their varied formation but are widely detected across many cellular proteins. Carbonylation prominently expresses itself in intracellular and extracellular proteins affecting many tissues and organs. Laboratories often use the DNPH (dinitrophenylhydrazine) assay and other protein carbonylation assays to detect and quantify these modifications.
Biological function summary
Protein carbonylation alters protein function and structure. This process disrupts protein-protein interactions and impairs enzyme activity leading to protein dysfunction. Carbonylated proteins fail to perform effectively within cellular systems and accumulate in cells. These accumulations can form large aggregates which cells often recognize as damaged. Protein degradation systems struggle to efficiently degrade these aggregates which can lead to further cellular dysfunction.
Pathways
Protein carbonylation integrates into the oxidative stress response pathways. In particular it affects the proteostasis network by challenging the protein quality control systems which include chaperones and proteasomes. This can influence pathways like the ubiquitin-proteasome pathway as proteins like ubiquitin tag damaged proteins for degradation. Additionally carbonylated proteins intertwined with inflammatory pathways interact with proteins such as nuclear factor kappa B (NF-kB) contributing to pro-inflammatory signaling.
Protein carbonyls associate closely with neurodegenerative diseases like Alzheimer's disease and Parkinson's disease. The increase in oxidative stress in these conditions leads to the accumulation of carbonylated proteins which correlate with disease progression. In Alzheimer's disease oxidative damage results in the carbonylation of the amyloid precursor protein enhancing amyloid-beta accumulation. In Parkinson’s disease alpha-synuclein undergoes carbonylation promoting its aggregation into Lewy bodies. These protein associations highlight the significant role of protein carbonyls in disease pathology.
