Oxidative stress has been implicated in the pathogenesis of neurodegenerative disorders, cancer, and aging. Oxidative stress occurs as a response to increased oxidants, decreased anti-oxidants, or failure to repair oxidative damage induced by reactive oxygen species (ROS). ROS are free radicals, reactive molecules containing oxygen, or molecules containing oxygen that generate free radicals. ROS include nitric oxide (NO), superoxide (O₂^-), peroxynitrite (ONOO^-), and hydroxyl radical (OH^-). ROS are cleared from cells by the action of superoxide dismutase (SOD), catalase and peroxidases.

NO, produced by nitric oxide synthases (NOS), is a critical signaling molecule involved in many physiological processes including muscle contraction, vasodilation, neuronal transmission, and immune responses. Some of these effects are mediated through reaction with and activation of guanylate cyclase (GC), resulting in increased cGMP production. With a half-life of seconds, NO readily diffuses through the cytosol and cell membrane, allowing it to act inside or adjacent to the cell in which it was produced.

ROS may interact to form agents that modify cellular proteins. Superoxide, produced by NADPH oxidase (phox), reacts with NO to form peroxynitrite. Intracellular peroxynitrite can modify proteins by interacting with and nitrating tyrosine residues to form 3-nitrotyrosine. Tyrosine nitration may increase (e.g. sGC, Src, PI3K, Akt), decrease (e.g. Mn-SOD, Ca⁺⁺-ATPase), or have no discernable effect (e.g. p53, VASP, α-Synuclein) on the activity of a particular protein.

Upstate′s new Nitrotyrosine Assay Kit, Chemiluminescence Detection (Cat. # 17-376) is a competitive ELISA for the quantitation of tyrosine nitration. The kit includes all reagents needed for measurement of tyrosine nitration, including white high binding 96-well plates, nitrated BSA standard, a Nitrotyrosine antibody, LumiGLO chemiluminescent detection substrate, and wash buffers.

The assay has a wide dynamic range and high precision, making this assay a valuable new tool for the study of tyrosine nitration.

White high binding plates. Two Costar^® 3922 white high-binding 96-well plates. TBS, 20X. Catalog # 20-190C. One vial containing 100 mL of 1M Tris, 3M^™ NaCl, pH 7.4. 20% TWEEN^®-20 (v/v). Catalog # 20-246. One vial containing 3 mL of 20% TWEEN^®-20 in sterile, distilled water. Blocking Buffer 2, 10X. Catalog # 20-316. One vial containing 7.5 mL of a proprietary buffer formulation, 10X stock containing 0.05% Kathon^®. Anti-Nitrotyrosine, 200X. Catalog # 07-752. One vial containing immuno-affinity purified rabbit IgG in 25mM Tris, pH 7.4, 137mM sodium chloride, 2.7mM potassium chloride, 0.05% Kathon^® and 5mg/ml BSA, 200X stock. Nitrated BSA. Catalog # 13-127. One vial containing 1 mg in 200 mL of nitrated BSA in 25mM Tris, pH 7.4, 137mM sodium chloride, 2.7mM potassium chloride. The 3-nitrotyrosine content was determined spectrophotometrically (ξ 438 =4300 M-1 cm-1) to be 5.6 mol nitrotyrosine per mol BSA. Goat Anti-Rabbit IgG, HRP-conjugate. Catalog # 12-348-MN. One vial containing 25 μg goat anti-rabbit IgG conjugated to horseradish peroxidase, in 25 μL 0.02M potassium phosphate, 0.15M NaCl, pH 7.2, 10 mg/mL BSA, 0.01% (w/v) gentamicin sulfate before the addition of glycerol to 50%. LumiGLO Chemiluminescent Substrate. Reagent A. Catalog # 20-212-c. One vial containing 10 mL of substrate. LumiGLO Chemiluminescent Substrate. Reagent B. Catalog # 20-212-d. One vial containing 10 mL of substrate.

3M is a trademark of 3M Company

Costar is a registered trademark of Corning, Inc.

Kathon is a registered trademark of Rohm and Haas Co.

TWEEN is a registered trademark of Croda International PLC

Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.