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The useful kit for aging, oxidative stress and various oxidative stress-related disease research

Protein Carbonyls Western Blot Detection Kit


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Background

Reactive oxygen species (ROS) which produced into the cells and tissues oxidize many proteins nonspecifically. Protein carbonyls are well-known to typical oxidized proteins which formed amino acyl carbonyl groups into the amino acids such as Lysine, Arginine, Proline and Threonine by ROS. Since carbonyl derivatives are chemically stable, it can use as a conventional oxidative stress marker.
In special features of this kit, this kit used 2,4-Dinitrophenylhydrazine (DNPH) for the derivative of carbonyl groups and specifically detect DNP-conjugate forms by using anti-DNP antibody. In the previous old methods, each sample has to pre-derivatize by DNPH before electrophoresis. And it was very difficult to identify individual protein carbonyls because pre-derivative alters protein charge resulting in different electrophoresis mobility. To avoid this problem, this kit derivatizes protein carbonyls to DNP-conjugate form onto the transferred membrane. That is, it is not necessary to pre-derivatize each sample before electrophoresis. This simple operation contributes to much shorter experimental time in the laboratory. This kit can detect specifically oxidized proteins.


 Oxidative Stress and Aging Research Kits Flyer [PDF]

Kit component

Assay 15 Blots (7.5 cm X 8.5 cm)
Antibody Rabbit anti-DNP antibody@0.075 mL (10 mM Tris (pH 7.6), 0.14 M NaCl)
šThis kit does not contain NaN3
DNPH solution 10X 2,4-Dinitrophenylhydrazine (DNPH) solution@15 mL(shade the light)
Oxidized protein oxidized BSA , soluble in SDS-PAGE sample buffer @0.15 mL
Storage and Stability antibody, DNPH solution, oxidized protein  4°C, 1 year

<Western Blot Analysis>
šDNP antibody at 1:2,000 dilution used
DNPH(+) F DNPH in 2 N HCl
Left lane:oxidized BSA 0.1 µg
Right lane:mouse liver extract 5 µg
DNPH(-) F 2 N HCl
Left lane:oxidized BSA 0.1 µg
Right lane:mouse liver extract 5 µg
CBB stainF proteins stained by Coomassie brilliant blue

šThe membrane was incubated with (DNPH(+)) or without (DNPH(-)) DNPH solution. Protein carbonyls were detected only DNPH-treated membrane but not detected at all untreated membrane. [Data from Dr. Akihito Ishigami, Tokyo Metropolitan Institute of Gerontology]

 

References

1. Nakamura A. et al., Analysis of protein carbonyls with 2,4-dinitrophenyl hydrazine and its antibodies by immunoblot in two-dimensional gel electrophoresis. J Biochem (Tokyo). 119 768-774 (1996)
2. Goto S. et al., Age-associated, oxidatively modified proteins: A critical evaluation. Age 20 81-89 (1997)
3. Goto S. et al., Carbonylated Proteins in Aging and Exercise: Immunoblot Approaches. Mech Ageing Dev 107 245-253 (1999)
4. Nakamura A. et al., Vitellogenin-6 is a major carbonylated protein in aged nematode, Caenorhabditis elegans. Biochem Biophys Res Commun. 264 580-583 (1999)
5. Robinson CE. et al., Determination of protein carbonyl groups by immunoblotting. Anal Biochem. 266 48-57 (1999)
6. Sato T. et al., Senescence marker protein-30 protects mice lungs from oxidative stress, aging, and smoking. Am J Respir Crit Care Med. 174 530-537 (2006)

Product List

Product Name Cat# Quantity Price

Protein carbonyls western blot detection kit(15Blots) (Rabbit)  DataSheet

SML-ROIK03-EX

1 KIT N/A

To be used for research only. DO NOT use for human gene therapy or clinical diagnosis.



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