RayBio^® Nitrated Alpha-synuclein (Tyr125/133) Quantitative ELISA Kit is an in vitro enzyme-linked immunosorbent assay for the quantitative measurement of Nitrated Alpha-synuclein (Tyr125/133). An anti-pan Alpha-synuclein antibody has been coated onto a 96-well plate. Standard and samples are pipetted into the wells and Alpha-synuclein present in a sample is bound to the wells by the immobilized antibody. The wells are washed, and rabbit anti-Nitrated Alpha-synuclein (Tyr125/133) antibody is used to detect only Nitrated protein. After washing away unbound antibody, HRP-conjugated anti-rabbit IgG is pipetted to the wells. The wells are again washed, a TMB substrate solution is added to the wells and color develops in proportion to the amount of Nitrated Alpha-synuclein bound. The Stop Solution changes the color from blue to yellow, and the intensity of the color is measured at 450 nm.

• Rapidly measures acetylated protein in lysates
• Screen numerous different cell lysates without performing a Western Blot analysis
• Minimal hands-on time, convenient, and non-radioactive material

• Anti-pan-Alpha-synuclein Microplate
• Nitrated Alpha-synuclein Standard
• Anti- Nitrated Alpha-synuclein (Tyr125/133) Antibody
• HRP-conjugated anti-rabbit IgG concentrate
• Wash Buffer
• Assay Diluent
• Lysis Buffer
• TMB One-Step Substrate Reagent
• Stop Solution

• 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

1. Prepare all reagents, samples, and Positive Control as instructed. in the manual.
2. Add 100 µL Standard to each well.
3. Incubate 2.5 hours at room temperature or overnight at 4ºC with gentle shaking.
4. Add 100 µL prepared detection antibody to each well.
5. Incubate for 1 hour at room temperature with gentle shaking.
6. Add 100 µL prepared HRP-conjugated anti-rabbit IgG concentrate solution.
7. Incubate for 1 hour at room temperature with gentle shaking.
8. Add 100 µL TMB One-Step Substrate Reagent to each well.
9. Incubate 30 minutes at room temperature.
10. Add 50 µL Stop Solution to each well.
11. 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.