Biotin Switch Assay Kit (S-Nitrosylation) (ab236207)
Key features and details
- Sample type: Adherent cells, Suspension cells, Tissue
Overview
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Product name
Biotin Switch Assay Kit (S-Nitrosylation)
See all Nitric oxide kits -
Sample type
Tissue, Adherent cells, Suspension cells -
Product overview
Biotin Switch Assay Kit (S-Nitrosylation) (ab236207) uses a modification of the Jaffrey et al. 'Biotin-switch' method to measure S-Nitrosylated (S-NO) proteins in whole cells or tissues by fluororescent microscopy, or in cell and tissue lysates by western blotting.
In the biotin switch assay method:
- firstly free SH groups are blocked (adding 125.1 amu per residue)
- then any S-NO bonds present as nitrosylated cysteines in the sample are cleaved
- the newly formed SH groups are then biotinylated (adding 523.6 amu per residue)
- the degree of biotinylation is then detected using either FITC-conjugated streptavidin for fluorometric detection, or HRP-conjugated streptavidin for colorimetric detection, and either western blotting or microscopy -
Notes
Nitric oxide (NO) is produced by three distinct isoforms of nitric oxide synthase and functions as a key signaling molecule in physiology and pathophysiology. NO transduces its effects by reacting either directly with heme and non-
heme centers of proteins or indirectly via further oxidation to various reactive nitrogen species (RNS).
Properties
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Storage instructions
Please refer to protocols. -
Components 1 kit S-Nitrosylation Blocking Reagent 3 vials S-Nitrosylation Buffer A 1 x 30ml S-Nitrosylation Buffer B (5X) 1 x 60ml S-Nitrosylation Detection Reagent I (HRP) 1 vial S-Nitrosylation Detection Reagent II (Fluorescein) 1 vial S-Nitrosylation Labeling Reagent 3 vials S-Nitrosylation Reducing Reagent 3 vials S-Nitrosylation Wash Buffer (10X) 1 x 25ml -
Research areas
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Relevance
Nitric oxide (NO) is a key vertebrate biological messenger, playing an important role in neurotransmission, vascular regulation, immune responses and apoptosis. NO , also known as "endothelium-derived relaxing factor" or "EDRF", is synthesized from L-arginine, oxygen and NADPH by various NO synthases. Most of the NO in the cell is oxidized to nitrite (NO2-) and nitrate (NO3-), and therefore the concentrations of these anions are generally as a quantitative measure of NO production.
Images
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Derivitization of protein nitrosothiol (S-NO) with biotin
Protein free thiols are derivatized with a blocking agent. S-Nitrosothiols are reduced to yield free thiol(s) which are covalently labeled with maleimide-biotin. Subsequent detection by avidin-coupled reagents can be used to localize the biotinylated protein(s).
