ABTS diammonium salt (AzBTS-(NH4)2) is used as a substrate for horseradish peroxidase (HRP) conjugate.

Instructions
I. Solution preparation
1. Mother solution preparation: ABTS diammonium salt is dissolved in water, usually distilled water or deionized water. The concentration is generally 1–10 mM, and the specific concentration depends on the experimental requirements.
II. Operation steps
1. Sample preparation: The enzyme substrate to be detected is generally a solution containing horseradish peroxidase (HRP). ABTS can react with hydrogen peroxide catalyzed by HRP to generate a colored product.
2. Reaction conditions:
Reaction buffer: It is recommended to use a buffer in the pH range of 3.5 to 4.5 (such as citric acid buffer).
Reaction temperature: The reaction is usually carried out at room temperature, and the reaction time is 10-30 minutes.
Catalyst: Hydrogen peroxide (H₂O₂) is added to the ABTS solution as an oxidant, and HRP catalyzes the reaction to generate a colored product.
3. Product Detection: The resulting product (usually green) can be quantified for HRP activity by measuring absorbance at 414 nm using a UV-Vis spectrophotometer.
4. Product Isolation and Purification: If desired, the resulting product can be further purified by chromatography, especially when the ABTS reaction is used for bioanalysis.
5. For purification and characterization, peroxidase-positive transformants are grown on a large scale (XL) under conditions that produce active protein in the culture supernatant. After 160 h of culture, when the activity relative to the substrate ABTS reaches 55,000 U/L, the supernatant containing peroxidase is collected. With ABTS as substrate, peroxidase activity decreases significantly when H2O2 concentrations rise above 0.125 mM, indicating that the enzyme is inhibited by H2O2. Depending on the substrate measured, the maximum reaction rate can reach 31.2 to 125 μM.