Nitric oxide (NO) is a reactive radical species, it is produced by NO synthases (NOS), a family of enzymes encoded by three distinct genes. NO participates in diverse biological process and physiological regulation including gastrointestinal, respiratory, cardiovascular and host defense. These various bioactivities result from the covalent adduction of an NO group to a cysteine thiol of target protein. This process is called S-nitrosylation, a redox-based, reversible, post-translational modification that regulates diverse signaling pathways and implicated in disease states.

RayBio® S-Nitrosylation Detection Kit uses a modified 'biotin-switch' method (Jaffrey et al.) to allow for the direct visualization of S-nitrosylated proteins by western blot analysis. In this S-nitrosylation biotin switch assay, unmodified free cysteines are first blocked. S-nitrosylated cysteines are then selectively reduced for specific labeling with biotin-maleimide reagents, which irreversibly bind to the cysteine thiol that was S-nitrosylated. Biotinylation of the newly formed thiol groups can then be detected by western blot. In addition, avidin resin can be used to selectively enrich S-nitrosylated proteins/peptides labeled with biotin. This procedure also allows for S-nitrosylation site mapping by using mass spectrometry.

1. Acetone, ≥ 98% (hazardous)
2. 1.5 ml microcentrifuge tubes
3. 15 ml tubes (polypropylene)
4. 10 ml graduated cylinders (X2)
5. Benchtop centrifuge and microcentrifuge (4°C)
6. Water bath or heat block
7. Distilled or deionized water
8. Precision pipettes to deliver 2 µl to 1 ml volumes
9. Adjustable 1-25 ml pipettes for reagent preparation

1. Prepare all reagents and samples as instructed.
2. Block unmodified free cysteines using 200 µl prepared S-Nitrosylation Blocking Buffer at 50°C with gentle rocking.
3. Precipitate proteins with ice-cold acetone.
4. Wash protein pellet twice with cold acetone/water mixture.
5. Resuspend protein pellets in Buffer B containing Reducing and Labeling Reagents.
6. Precipitate proteins with ice-cold acetone.
7. Resuspend protein pellets.