The addition of sulfuric acid stop solution changes the color to yellow at 450 nm using a spectrophotometer or microplate reader Accurate strength measurement. This specific formulation is used in most Thermo Scientific Pierce ELISA kits to provide consistent performance for sandwich ELISA technology.

TMB Substrate Solution Features:

•Product Number:N301
• Specification:100 mL (enough for Approximately 10 96-well microplates)
• Storage: Ship on a cold bag. Store immediately upon receipt at 2-8°C. Do not use products past their expiry date.
•Applications: ELISA: Recommended for peroxidase-based immunoassays. Upon oxidation, TMB forms a water-soluble blue reaction product that can be measured spectrophotometrically at 605 nm. After acidification, the reaction product turned yellow and the absorbance peak was located at 450 nm.
•Note: This product is sensitive to contamination from various oxidants. To avoid contamination and premature failure, avoid exposing TMB solutions to any potential sources of contamination. Never pipette directly from the bottle. Pour the required amount into the test tube and pipet from the tube. Do not pour excess TMB back into the primary storage container.

Procedure:
•Add 100 μL TMB substrate to each well containing the peroxidase reaction solution.
•Incubate at room temperature for 15 to 30 minutes.
•Add 100 μL of stop solution (0.16 M sulfuric acid, N600) to each well.
•Measure absorbance at 450 nm.