TMB Substrate Solution ‘Ready to Use’

The high quality of the substrate can be preserved by storing at temperatures between 2 – 8ºC. The substrate should not be frozen and should be protected from direct light by storing in amber bottles. Do Not Freeze

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Do not use this product beyond the stated expiration date on the product label. <br> <b>Precautions:</b> 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, such as metals or silica based materials. Never pipette directly from the bottle. Pour out the required amount onto a tube and pipette from the tube. Only high quality glass and plastic products should be used for storing aliquots. Do not return excess TMB to the primary storage container.

TMB Microwell Substrate (3,3’,5,5’ tetramethylbenzidine) is a soluble substrate used with the enzyme horseradish peroxidase (HRPO) designed for various qualitative or quantitative immunoassays but not recommended for membrane or immunohistochemical applications where a precipitating reaction product is required. Initially, the substrate should be colorless or slightly yellow in color and will be stored in a mildly acidic buffer. TMB Microwell Substrate turns a deep blue color when oxidized with hydrogen peroxide catalyzed with horseradish peroxidase labeled conjugates with absorbencies at 370 nm or in a range of 620 nm to 650 nm. The color is changed to a bright yellow if an acidic stop reagent such as HCl or sulfuric acid is used. The absorbance should be read at 450 nm if the reaction is stopped which increases the sensitivity 2-4 fold.

Leinco Technologies’ TMB Microwell Substrates exhibit superior kinetic performance, sensitivity and lot to lot consistency as compared to other vendors. The outstanding shelf life of at least forty eight months for the TMB Microwell Substrate makes this reagent ideal for long term use of the same manufacturing lot.

Leinco Technologies offers a diverse selection of high-performance TMB substrates engineered to elevate your ELISA assays. Upgrade your ELISA workflow with Leinco's TMB substrates and experience the difference in performance and reliability.

TMB Microwell Substrate is a ready to use solution that needs no preparation or dilution (Note: Allow the substrate to equilibrate at room temperature (~25^°C) prior to use). Pour estimated amount of substrate into a suitable high quality plastic reservoir to avoid contamination of the bulk solution. Again, it is recommended that you allow the substrate solution to equilibrate to room temperature before use. While the TMB solution is equilibrating, wash the microplates thoroughly to remove excess peroxidase labeled conjugates. Washing the plates at least four times is recommended to minimize background noise.

End-Point Assays:
1.) Add 100 μl of substrate solution to each well of a 96 well ELISA plate.
2.) Allow the substrate reaction to develop and wait for a soluble blue reaction product to develop(Note: This typically takes 15-20 minutes).
3.) The reaction should be stopped by using a 450 nm or 650 nm stop solution. (Note: A soluble yellow product develops after stopping and stabilizing with a 450 nm stop solution.)
4.) Measure the absorbance at 450 nm or 650 nm of each microwell within 1 hour.

Kinetic Assays: 
1) Add 100 µL of equilibrated substrate solution to each microtiter plate well. 
2) Measure the absorbance at 650 nm at multiple time points.
3) Calculate the rate of absorbance change for each microwell. 

 

Leinco is a leading USA manufacturer, located in St. Louis, Missouri, of premium immunoassay reagents, committed to advancing disease diagnostics and treatment. Our ISO 13485-certified facility adheres to the highest quality standards, ensuring consistent and reliable performance for your immunoassay development. Our comprehensive suite of high-quality reagents empowers assay developers to build consistent, reliable, and sensitive immunoassays for a wide range of applications, including ELISA, point-of-care diagnostics, and beyond.