TMB, Soluble

Peroxidase substrate that produces a blue soluble end product that can be read at 370 nm or 650 nm. Use of stop solution enhances sensitivity two- to four-fold and results in a yellow solution that can be read at 450 nm.

Soluble TMB substrate supplied as a single component system.

Toxicity: Irritant (B)

Single component system containing 1.46 mM TMB, 2.21 mM H₂O₂ in a proprietary solvent, pH 3.1 ± 0.5.

Protect from exposure to direct sunlight. Discard if the solution turns blue or turbid. The reagent is stable at room temperature, but we recommend storage in the refrigerator for longer shelf life. Warm to assay temperature before use.

Product tested for stability at 4°C and 18-26°C. Product performance is tested on the final product by ELISA.



Suggested Procedure for Use of TMB in HRP-based ELISAs:



1. Complete all required incubations with antibodies and HRP-labeled probes.

2. Wash plate wells at least 4 times with phosphate-buffered saline (PBS) or Tris-buffered saline (TBS), containing 0.1% Tween^®-20 detergent.

3. After the final wash, shake and blot all residual buffer from the wells.

4. Add 100 μl TMB solution to each well and incubate for 5-30 min.

5. Options for measurements:

a. For kinetic assays, the reaction can be monitored as a function of time by reading absorbance at 650 nm at intermediate intervals.

b. For endpoint assays that preserve the blue chromogen, the reaction should be stopped by addition of 100 µl of 0.1% sodium fluoride (NaF) and the absorbance read at 650 nm.

c. If increased sensitivity is desired for endpoint assays, the reaction should be stopped by addition of 100 μl of either 500 mM H₂SO₄ or 250 mM HCl and the absorbance read at 450 nm WITHIN 5 MIN. Addition of acid converts the blue radical to the yellow diimine, which absorbs at 450 nm.



NOTE: Optimal incubation times may vary depending on the amount of HRP present. If color develops too quickly, zero-order kinetics will not prevail. Dilution of the probe, antibody, or HRP-labeled reagent may be required. Variations in time, reagent volumes, and temperature may also require further standardization by the user.

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