TMB Substrate Buffer is a single component, ready-to-use, working solution recommended for ELISA (microwell) procedures. TMB (3, 3′, 5, 5′-tetramethylbenzidine) is a commonly used substrate for the enzyme horseradish peroxidase (HRP) and the combination is one of the most acknowledged ELISA detection systems. Develops a soluble blue reaction product that may be read at 655nm. For endpoint assays the reaction may be stopped with acid, forming a yellow reaction product that may be read at 450nm or 405nm. Not recommended for membrane applications.
• This substrate is very light sensitive and must be protected from direct sunlight or any UV source.
• Bring to room temperature (approx. 25°C) before use. Note: Dilution of the substrate is not recommended. To reduce the intensity of a reaction, it is recommended that the antibodies or conjugates be diluted.
• Add 100 μl of TMB Substrate Buffer to each well and incubate for 5 –10 minutes at room temperature.
• A blue colored reaction product will develop, and negative control reactions remain clear non-colored solution in the wells.
• Add 100 μl of 1N H2SO4 to stop enzyme reaction after 10 minutes incubation. The blue color solution will turn to a yellow color.
•Blue reaction product, read at 655nm. Yellow, read at 450nm.
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