Nitrotyrosine Competititive ELISA Kit: 96 Tests

NWLSS | Northwest Life Science Specialties, LLC.
NWK-NTR02-01 $775.00 each

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The Competitive Nitrotyrosine (NTR) ELISA is recommended for the detection of nitrosylated proteins in plasma or serum samples. Designed as an improved method for measuring monosubstituted NTR-protein adducts, this ELISA has been optimized around a standard with a single NTR substitution. In contrast, our original sandwich version NTR ELISA employs a standard with two or more substitutions per molecule. While both methods detect nitrotyrosine protein adducts, it is expected that modifications due to lower level nitrosative stress may be more easily detected using the new competitive ELISA

Introduction

Nitrate (NO3) and nitrite (NO2) production are typically used as markers of nitric oxide production (NO) production. These analytes by themselves fail to address the eventual fate of NO or the possible adverse effects associated with its excess production and reaction with free radical species in vivo. Active NO metabolites can react with superoxide to form peroxynitrite (ONOO-) a powerful oxidant and nitrating agent. Subsequent reaction of peroxynitrite with proteins results in nitrotyrosine formation.

As a stable end product of peroxynitrite mediated oxidation/nitration, Nitrotyrosine can be used as a surrogate index of NO dependent damage in vivo and has been associated with multiple disease states.

Test Principle

This assay features a “competitive” ELISA format in which standards and samples are first incubated with a biotynylated Tracer Antibody in a separate U-bottom microplate. After incubation, the standards and samples are transferred to a microplate precoated with nitrated-HSA and allowed to incubate for an additional hour.

During this step unbound Biotinylated Tracer Antibody binds to stationary phase NO-HAS. A subsequent wash removes excess Tracer and Tracer/protein complex. Addition of streptavidin-peroxidase followed by tetramethylbenzidine (TMB) facilitates color development inversely proportional to the nitrotyrosine present in the sample. The reaction is stopped using an oxalic acid solution and the assay is read on a plate reader at 450 nm.

Does the NWK-NTR02-01 ELISA detect free nitrotyrosine or nitrosylated protein adducts?

The NWK-NTR02-01 ELISA recognizes nitrotyrosine-protein adducts. It is capable of detecting mononitrated proteins.

How should tissues be homogenized for assay using the NWK-NTR02-01 kit?

We normally recommend that tissue samples re homogenized in normal PBS, pH 7.0. Homogenization can be performed with a dounce style or Potter Elvehjem style tissue grinder or similar apparatus. Tissue homogenates should be centrifuged and the clarified supernatant tested for nitrotyrosine. Several references wherein other researchers have used the NWK-NTR01 kit to test tissue samples can be found in the citations references for this product.

For Research Use Only

Catalog Number: NWK-NTR02-01
Format: 96 wells presented as 1 plate sectionable as 8 well individual strips
Sample Requirements: Plasma, serum and other biological samples such as tissue homogenate samples.
Specificity: Nitrosylated protein adducts
Sensitivity: 50 nM monovalent nitrotyrosine (single modification per molecule)
Storage and Stability: 1 year from manufacture date when stored at specified temperature
Kit Contents: Precoted Microwell strips and strip frames
Wash Buffer
Dilution Buffer
Nitrotyrosine Standard
Biotinylated anti nitrotyrosine tracer
Streptavidin Peroxidase
TMB Substrate
Stop Solution

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