TMB HRP Microwell Substrate UltraSensitive™ One Component

TMB HRP Microwell Substrate UltraSensitive™ One Component

Product No.: T263

[product_table name="All Top" skus="T263"]

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Product Type
Substrate and Assay Reagents

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Product Size
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Product Details

Storage and Handling
Store at temperatures between 2-8ºC. Do Not Freeze. The substrate should be protected from direct light by storing in amber bottles. Only high quality amber glass and plastic products should be used for storing aliquots.
Expiration Date
Stable for a minimum of 30 months from the manufactured date when properly stored between 2-8ºC.


TMB HRPO Microwell UltraSensitiveTM Substrate (3,3’,5,5’ tetramethylbenzidine) is a soluble substrate used with the enzyme horseradish peroxidase (HRPO) designed for various qualitative and 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 HRPO Microwell UltraSensitiveTM Substrate turns a deep blue color when reacted 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 thirty months for the TMB HRPO Microwell UltraSensitiveTM Substrate makes this reagent ideal for long term use of the same manufacturing lot.
Directions for Use
TMB HRPO Microwell UltraSensitiveTM Substrate is a ready to use solution that needs no preparation or dilution. Pour estimated amount of substrate into a suitable high quality plastic reservoir to avoid contamination of the bulk solution. 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.

Add 100 μl of substrate solution to each well of a 96 well ELISA plate. Once a soluble blue reaction product develops, the plate can be read at 370 nm or in the range of 620 nm to 650 nm. The absorbance values of the sample should be monitored so that a linear curve can be plotted. For increased sensitivity, add 50 μl of a stop solution such as 1.0 N HCl or 1.0 N sulfuric acid to create a soluble bright yellow reaction product. After stopping the enzymatic reaction the plate should be read at 450 nm. Estimated incubation times for substrate range from 15 to 25 minutes.
Problem Possible Causes Possible Solutions
High Background Noise Insufficient plate washing · Increase number of washes
· Add detergent or protein to wash solution
· Allow a short soak period between washes
Concentration of enzyme is too high · Check calculations and titrate if necessary
High incubation time  · Reduce incubation time
Contaminated buffers or solutions  · Repeat assay with fresh buffers and solutions
No/Low Signal Capture antibody did not bind to plate · Evaluate coating conditions and standardize
· Increase coating time
 · Increase coating concentration
· Change plate type to high binding
Contaminated buffers or incorrect solutions  · Repeat assay with fresh buffers and solutions
Not enough reporter antibody used  · Increase concentration of HRPO labeled antibody
Poor assay-to-assay reproducibility Inconsistent washing  · Standardize washing and ensure thoroughness
Variations in incubation temperature or time · Ensure constant temperature and incubation time during incubations
· Ensure all reagents are at constant temperature when added
Low reading across entire plate Incorrect wavelength on plate reader · Check filters
· Check absorbance wavelength
Insufficient development time  · Increase development time until background appears

Related Protocols

Elisa Sandwich Protocol
Products are for research use only. Not for use in diagnostic or therapeutic procedures.