Nitrotyrosine ELISA Kit

Sample

cell lysates, serum, plasma and purified proteins

Detection Method

cELISA

Intended Use

The CD Nitrotyrosine ELISA Kit is a competitive enzyme immunoassay developed for rapid detection and quantitation of 3-nitrotyrosine in protein sample. The quantity of 3-nitrotyrosine in protein sample is determined by comparing its absorbance with that of a known nitrated BSA standard curve. The kit has a nitrotyrosine detection sensitivity range of 20 nM to 8.0 μM. Each kit provides sufficient reagents to perform up to 96 assays, including standard curve and unknown protein samples.
FOR RESEARCH USE ONLY. Not for use in diagnostic procedures.

Contents of Kit

1. Nitrotyrosine Coated EIA Plate: One strip well 96-well plate.
2. Anti-Nitrotyrosine Antibody: One 20 μL vial of anti-nitrotyrosine Rabbit IgG.
3. Secondary Antibody, HRP Conjugate: One 20 μL vial.
4. Assay Diluent: One 50 mL bottle.
5. 10× Wash Buffer: One 100 mL bottle.
6. Substrate Solution: One 12 mL amber bottle.
7. Stop Solution: One 12 mL bottle.
8. Nitrated BSA Standard: One 500 μL vial of 1 mg/mL Nitrated BSA in PBS with a nitrotyrosine content of 40 μM (2.7 mole of nitrotyrosine per mole of BSA). The protein nitrotyrosine level is predetermined by a spectrophotometric method as described by Ischiropoulos et al.

Storage

Upon receipt, aliquot and store the Nitrated BSA Standard at -20°C to avoid multiple freeze/thaw cycles. Store all other kit components at 4°C.

General Description

The modification of tyrosine residues in proteins to 3-nitrotyrosine by peroxynitrite or other potential nitrating agents has been detected in biological systems that are subject to oxidative stress. Detection of nitrotyrosine-containing proteins has been reported in many human and animal diseases or cellular models of disease. While all tyrosine residues in proteins may theoretically be targets for nitration, presumably the efficiency of tyrosine nitration is dependent on various biological conditions such as the local production and concentration of the reactive species, the existence and availability of antioxidants and scavengers, the accumulation of inflammatory cell and the presence of proinflammatory cytokines, as well as the proximity and compartmentation of these components.

Standard Curve

The following figures demonstrate typical Nitrotyrosine ELISA results. One should use the data below for reference only. This data should not be used to interpret actual results.
Nitrotyrosine ELISA Standard Curve:

Protein Nitration by tetranitromethane. STO (MEF) cells were lysed in 25mM HEPES, pH 7.5, 150 mM NaCl, 1% NP-40, 10 mM MgCl₂, 1 mM EDTA, 2% Glycerol. Cell Lysate was nitrated with tetranitromethane (TNM). The protein 3-nitrotyrsone levels were determined as described in the assay instructions.