Description
Key facts
Sample types:Suspension cells, Tissue Homogenate, Adherent cells
Product details:
Protein Carbonyl Assay Kit (Western Blot) ab178020 is designed for the measurement of protein carbonyl groups that are created by the oxidation of proteins. This can be through reaction with ozone or oxides of nitrogen, or by metal catalyzed oxidation.
In the protein carbonyl assay protocol, carbonyl groups in protein side chains are derivatized to DNP-hydrazone by reaction with DNPH. The DNP moieties are detected using an anti-DNP antibody and western blotting.
Using an immunoblotting method for the protein carbonyl assay has the advantage that individual oxidized proteins are separated and identified from a complex mixture by SDS-PAGE. The oxidative status of each protein can be compared between samples.
Protein carbonyl assay protocol summary:
- extract proteins from samples with extraction buffer, 20 min incubation, centrifugation at 18,000 x g for 20 min, and retention of the supernatant
- denature proteins with SDS solution
- incubate with DNPH for 15 min
- add neutralization solution
- run samples on polyacrylamide gel
- transfer proteins to membrane
- block for 1 hr
- incubate with DNP antibody for 3 hr, and wash
- incubate with HRP goat anti-rabbit secondary antibody for 1 hr, and wash
- develop with ECL and acquire image
: RIPA buffer is not compatible with ab178020
Previously called Oxidized Protein Assay Kit (Western blot). Oxygen-derived free radicals have been implicated in important roles in aging, apoptosis, and cancer. These highly reactive chemical species are also involved in a wide variety of clinical disorders, such as atherosclerosis, cataractogenesis, neurodegenerative diseases, chronic inflammatory diseases, pulmonary diseases and cardiovascular diseases.
Properties and Storage Information:
Shipped at conditions-Dry Ice, Appropriate short-term storage conditions--20°C, Appropriate long-term storage conditions--20°C, Storage information--20°C
Supplementary Information:
This supplementary information is collated from multiple sources and compiled automatically.
Oxidized proteins arise when proteins undergo oxidative modifications especially through a process known as protein carbonylation. This involves the conversion of amino acid side chains into carbonyl derivatives such as aldehydes and ketones. Protein carbonylation serves as a marker for oxidative stress and carbonyl groups can be detected through methods like protein carbonylation assay and immunoblot assay sometimes referred to as oxyblot. These oxidized modifications do not have a defined mass as they vary depending on the protein and the extent of oxidation. Oxidized proteins are generally found in various cellular compartments and tissues and are often considered indicators of oxidative damage.
Biological function summary
Protein carbonylation represents a form of protein oxidation that can affect protein function and stability. It often occurs in response to oxidative stress and has implications in cellular signaling pathways. This modification can inhibit the normal activity of proteins and in some cases may lead to protein aggregation. Although oxidized proteins are not part of a specific complex excessive carbonylation may interfere with protein-protein interactions and disrupt cellular homeostasis. The accumulation of protein carbonyls is common during aging and in response to environmental stressors.
Pathways
Protein carbonylation interacts with signaling cascades that respond to oxidative stress. It plays a role in the oxidation-reduction process and intersects with the ubiquitin-proteasome pathway impacting protein degradation. Proteins like ubiquitin and the proteasome recognize carbonylated proteins as damaged targeting them for degradation. The presence of oxidized proteins can further affect other pathways such as apoptosis signaling due to their disruptive influence on cellular functions.
Protein carbonylation is associated with aging-related diseases and neurodegenerative disorders like Alzheimer's disease. Accumulation of oxidized proteins in neuronal cells contributes to the pathology of such diseases. Oxidized proteins interact with amyloid-beta an important protein in Alzheimer's pathology facilitating the progression of the disease. Additionally cardiovascular diseases also exhibit increased protein carbonylation suggesting a link to cardiac dysfunction. The modification of proteins such as low-density lipoprotein (LDL) by oxidative processes underlines the association between oxidized proteins and these conditions.
