Nori Human NOX2 ELISA Kit Summary

Alternative names for NOX2: NADPH oxidase 2, cytochrome b(558) subunit beta, Cytochrome b-245 heavy chain, CYBB

Standard Curve

Kit Components
1. Pre-coated 96-well Microplate
2. Biotinylated Detection Antibody
3. Streptavidin-HRP Conjugate
4. Lyophilized Standards
5. TMB One-Step Substrate
6. Stop Solution
7. 20 x PBS
8. Assay Buffer

Other Materials Required but not Provided:
1. Microplate Reader capable of measuring absorption at 450 nm
2. Log-log graph paper or computer and software for ELISA data analysis
3. Precision pipettes (1-1000 µl)
4. Multi-channel pipettes (300 µl)
5. Distilled or deionized water

Protocol Outline
1. Prepare all reagents, samples and standards as instructed in the datasheet.
2. Add 100 µl of Standard or samples to each well and incubate 1 h at RT.
3. Add 100 µl of Working Detection Antibody to each well and incubate 1 h at RT.
4. Add 100 µl of Working Streptavidin-HRP to each well and incubate 20 min at RT.
5. Add 100 µl of Substrate to each well and incubate 5-30 min at RT.
6. Add 50 µl of Stop Solution to each well and read at 450 nm immediately.

Background:

NADPH oxidase 2 (Nox2), also known as cytochrome b(558) subunit beta or Cytochrome b-245 heavy chain, is a protein that is encoded by the NOX2 gene (also called CYBB gene).^[1] The protein is a super-oxide generating enzyme which forms reactive oxygen species (ROS). Nox2 is composed of cytochrome b alpha (CYBA) and beta (CYBB) chain and contains an N-terminal transmembrane domain that binds two heme groups, and a C-terminal domain that is able to bind to FAD and NADPH.^[2] Nox2 is the catalytic, membrane-bound subunit of NADPH oxidase. It is inactive until it binds to the membrane-anchored p22phox, forming the heterodimer known as flavocytochrome b558.^[3] After activation, the regulatory subunits p67phox, p47phox, p40phox and a GTPase, typically Rac, are recruited to the complex to form NADPH oxidase on the plasma membrane or phagosomal membrane.^[4]  Nox2 may play an important role in atherosclerotic lesion development in the aortic arch, thoracic, and abdominal aorta.^[5][6] Nox2 may play a role in determining the size of a myocardial infarction due to its connection to ROS, which play a role in myocardial reperfusion injury. This was a result of the relation between Nox2 and signaling necessary for neutrophil recruitment.^[7] Furthermore, it increases global post-reperfusion oxidative stress, likely due to decreased STAT3 and Erk phosphorylation.^[7] In addition, it appears that hippocampal oxidative stress is increased in septic animals due to the actions of Nox2.^[8] Nox2 may also plays an important role in angiotensin II-mediated inward remodelling in cerebral arterioles due to the emittance of superoxides from Nox2-containing NADPH oxidases.^[9] CYBB transcript levels are upregulated in the lung parenchyma of smokers. ^[10]

References

1.      Royer-Pokora,B., et al. (1986) Nature 322 (6074), 32-38.

2.      Aguirre, Jesús; Lambeth, J (2010). Free Radical Biology and Medicine. 49 (9): 1342–1353.

3. Hervé C, et al. (2006). Current Genetics. 49(3): 190–204.
4. Kawahara T, Lambeth JD (2007). BMC Evolutionary Biology. 7: 178.
5. Sorescu, D; et al. (2002). Circulation. 105 (12): 1429–35.
6. Chaubey, S; et al. (2013). PLOS ONE. 8 (2): e54869. doi:1371/journal.pone.0054869.
7. Braunersreuther V, et al. (2013). Journal of Molecular and Cellular Cardiology. 64: 99–107.
8. Hernandes MS, et al. (2014). J Neuroinflammation. 11 (1): 36. doi:1186/1742-2094-11-36.
9. Chan SL, Baumbach GL (2013). Frontiers in Physiology. 4: 133.
10. Pintarelli G, et al. (2019). Scientific Reports. 9 (1): 13039. doi:1038/s41598-019-49648-2.