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protein carbonyl in specimen (OxyBlot assay)
Procedure:
- carbonyl groups on the protein side chains are derivatized to 2,4-dinitrophenylhydrazone (DNP-hydrazone) by reaction with 2,4-dintrophenylhydrazine
- the DNPH-derivatized proteins are separated by polyacrylamide gel electrophoresis (PAGE) followed by Western blotting
- alternatively, a dot-blot method can be used
- filter blots are then incubated with primary antibody (rabbit IgG) to DNP followed by incubation with a peroxidase-conjugated goat anti-rabbit IgG
- filters are then treated with chemiluminescent reagents (luminol & enhancer)
- light emission at 428 nm is detected on blue light-sensitive film after addition of hydrogen peroxide
Related
protein carbonyl
Specific
protein carbonyl in serum/plasma
General
special chemistry test
References
- OxyBlot (TM) Protein Oxidation Detection Kit,
Intergen Catalog # S7150
http://www.intergenco.com
- Climent I, Tsai L, Levine RL.
Derivatization of gamma-glutamyl semialdehyde residues in
oxidized proteins by fluoresceinamine.
Anal Biochem 182:226, 1989
PMID: 2575359