The RayBio® S-Nitrosylated NF-KB p65 ELISA Kit uses a modified 'biotin-switch' method to allow for the direct visualization of human and mouse S-Nitrosylated NF-KB p65 by ELISA. 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 ELISA. An anti-pan NF-KB p65 antibody has been coated onto a 96-well plate. Biotinylated samples are pipetted into the wells and NF-KB p65 present in a sample is bound to the wells by the immobilized antibody. The wells are washed and HRP-conjugated streptavidin is used to detect S-Nitrosylated NF-KB p65. The wells are again washed, a TMB substrate solution is added to the wells and color develops in proportion to the amount of S-Nitrosylated NF-KB p65 bound. The Stop Solution changes the color from blue to yellow, and the intensity of the color is measured at 450 nm.

• Quantify quickly and easily
• Screen numerous different lysates without performing a Western Blot analysis
• Minimal hands-on time, convenient, and non-radioactive material

• Acetone (pre-chilled (-20°C))
• Microplate reader capable of measuring absorbance at 450 nm
• Protease and Phosphatase inhibitors
• Precision pipettes to deliver 2 μL to 1 mL volumes
• Adjustable 1-25 mL pipettes for reagent preparation
• 100 mL and 1 liter graduated cylinders
• Log-log graph paper or computer and software for ELISA data analysis
• Absorbent paper
• Tubes to prepare positive control or sample dilutions
• Distilled or deionized water
• Shaker

1. Prepare all reagents, samples and Positive Control as instructed.
2. Block unmodified free cysteines using 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 containing Reducing and Labeling Reagents.
6. Precipitate proteins with ice-cold acetone.
7. Resuspend protein pellets.
8. Add 100 μL sample and Positive Control to each well. Incubate 2.5 hours at room temperature or overnight at 4ºC with gentle shaking.
9. Add 100 μL prepared HRP-Streptavidin solution. Incubate for 1 hour at room temperature with gentle shaking.
10. Add 100 μL TMB One-Step Substrate Reagent to each well. Incubate 30 minutes at room temperature.
11. Add 50 μL Stop Solution to each well. Read at 450 nm immediately.

The entire kit may be stored at -20°C for up to 6 months from the date of shipment. Avoid repeated freeze-thaw cycles.