Product nameProtein Carbonyl Assay Kit (Western Blot)
Sample typeAdherent cells, Suspension cells, Tissue Homogenate
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
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.
Review the oxidative stress marker and assay guide to learn about more assays for oxidative stress.
Storage instructionsPlease refer to protocols.
Components 100 tests 12%SDS 1 x 2ml 1X 2,4-Dinitrophenylhydrazine (DNPH) solution 1 x 4ml 1X Derivatization Control Solution 1 x 4ml 2X Extraction Buffer 1 x 4ml 5000X HRP Conjugated Scondary Antibody 1 x 100µl 5000X Primary anti-DNP Antibody 1 x 100µl Neutralization Solution 1 x 4ml Standard protein with DNP residues 1 x 1ml
Sensitivity of DNP detection demonstrated by serial dilution of DNP –derivatized BSA.
Lane 1: 5 ng BSA
Lane 2: 10 ng BSA
Lane 3: 20 ng BSA
Lane 4: 40 ng BSA
Lane 5: 80 ng BSA
Lane 6: 160 ng BSA
Example of DNP detection demonstrated by DNP derivatized H2O2 treated Hela cells.
Lane 1: MW ladder
Lane 2 (DNPH): HeLa cells with 0.1 mM H2O2, 15 minutes treatment.
Lane 3 (Negative Control): HeLa cells with 0.1 mM H2O2, 15 minutes treatment.
Lane 4 (DNPH): HeLa cells with 1 mM H2O2, 15 minutes treatment.
Lane 5 (Negative Control): HeLa cells with 1 mM H2O2, 15 minutes treatment.
Lane 6 (DNPH): HeLa cells with 10 mM H2O2, 15 minutes treatment.
Lane 7 (Negative Control): HeLa cells with 10 mM H2O2, 15 minutes treatment.
Lane 8 (DNPH): HeLa cells with 0 mM H2O2, 15 minutes treatment.
Lane 9 (Negative Control): HeLa cells with 0 mM H2O2, 15 minutes treatment.
Lane 10 (DNPH): BSA with DNPH derivatization.
Lane 11 (Negative Control): BSA with DNPH derivatization.
This product has been referenced in:
- Rajendran J et al. Alternative oxidase-mediated respiration prevents lethal mitochondrial cardiomyopathy. EMBO Mol Med 11:N/A (2019). Read more (PubMed: 30530468) »
- Nakazawa H et al. Coenzyme Q10 protects against burn-induced mitochondrial dysfunction and impaired insulin signaling in mouse skeletal muscle. FEBS Open Bio 9:348-363 (2019). Read more (PubMed: 30761259) »