Addition of sulfuric acid stop solution changes the color to yellow, enabling accurate measurement of the intensity at 450nm using a spectrophotometer or plate reader. This particular formulation is used in most of the Thermo Scientific Pierce ELISA Kits where it provides consistent performance for the sandwich ELISA technique.

Features of TMB Substrate Solution:

• Product Number: N301
• Size: 100 mL (sufficient for about ten 96-well microplates)
• Storage: Shipped on cold packs. Store at 2-8°C immediately upon receipt. Do not use product beyond the stated expiration date.
• Application: ELISA: Recommended for peroxidase-based enzyme immunoassays. Upon oxidation, TMB forms a water-soluble blue reaction product that can be measured spectrophotometrically at 605nm. Upon acidification, the reaction product becomes yellow with an absorbance peak at 450nm.
• Precautions: This product is sensitive to contamination from a variety of oxidizing agents. To avoid contamination and premature expiration, avoid contacting TMB solution with any potential source of contamination. Never pipette directly from the bottle. Pour out required amount onto a tube and pipette from the tube. Do not return excess TMB to primary storage container.

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