| Product Description | Aladdin's Supersensitive TMB Chromogen Solution for ELISA utilizes the single TMB chromogen solution to detect horseradish peroxidase (HRP) activity with low background production. This product can also be used to determine the content of HRP in samples such as blood or hemoglobin.TMB, also known as 3,3',5,5'-Tetramethylbenzidine, is a conventional substrate of HRP. Under the catalysis of HRP or other appropriate peroxidases, TMB produces a soluble blue reaction product which absorbs at 370nm or in a range of 620nm to 650nm [1,2]. The reaction can be terminated using an appropriate stop solution, such as Stop Solution for TMB Substrate (450nm, Sulfuric acid free) , Stop Solution for TMB Substrate (650nm, non-corrosiveness) , or 2M H2SO4. When using the Stop Solution for TMB Substrate (450nm, Sulfuric acid free) , a soluble yellow reaction product is produced and its absorbance can be measured at 450nm. However, when Stop Solution for TMB Substrate (650nm, non-corrosiveness) is used, a soluble blue reaction product with an absorbance at 620-650nm will be produced.This chromogen solution is a ready-to-use, single-component solution that does not require the addition of hydrogen peroxide or other reagents, simplifying the operation and ensuring more stable and reliable assay results.This chromogen solution is an aqueous solution, without any organic solvents such as alcohol, DMF, or DMSO, making it safe and non-toxic to use.This product is approximately 3 times more sensitive than Aladdin's TMB Chromogen Solution for ELISA , and enables a faster reaction rate as well. Please refer to Figure 1 and Figure 2 for the comparison of these two products.Figure 1. Reaction kinetics of Aladdin's Supersensitive TMB Chromogen Solution for ELISA and the TMB Chromogen Solution for ELISA . In 96-well plates, mix 20µl of diluted HRP-labeled Streptavidin in PBS, and 100µl of P0208 or P0209 in respective wells. The reactions were incubated at 25℃ and the absorbance was measured at 650nm every 2 minutes. As shown in the figure, the reaction rate of P0208 is approximately 3 times that of P0209. This figure is for reference only, which may vary due to different experimental conditions.Figure 2. The performance of Aladdin's Supersensitive TMB Chromogen Solution for ELISA and the TMB Chromogen Solution for ELISA in detecting the HRP-labeled Streptavidin . HRP-labeled Streptavidin was gradient-diluted with PBS. In 96-well plates, mix 20µl of diluted HRP-labeled Streptavidin and 100µl of P0208 or P0209 in respective wells, resulting in concentrations of HRP-labeled Streptavidin as shown in the figure. After incubating reactions at room temperature in the dark for 5 minutes, add 100µl of Stop Solution for TMB Substrate (450nm, sulfate-free) per well to stop the reactions, and absorbance at 450nm was measured. A, Standard curve with the concentration of HRP-labeled Streptavidin on the x-axis and A450 on the y-axis, and a four-parameter logistic regression (4PL) commonly used in ELISA was applied. B, Absorbance of reactions with low concentrations of HRP-labeled Streptavidin and a linear regression was performed. As shown in the figure, the signal value of P0208 reactions is approximately 3 times that of P0209 reactions with appropriate amounts of HRP. This figure is for reference only, which may vary due to different experimental conditions.This product is most commonly used for ELISA, but it can also be used to examine the peroxidase content in samples such as blood or hemoglobin.Typically, use 0.1mL of chromogen solution per well for ELISA. Thus per 100mL of this product is sufficient for approximately 1000 assays.
Precautions： Storage at -20℃ is not recommended for this product. The assay signal may decrease by approximately 10% after three freeze-thaw cycles.This product should be protected from direct light exposure. Avoid direct contact with heat sources, metals, or oxidizing agents.Do not use this product directly from the bottle. It is recommended to take out the required amount of solution from the bottle for each experiment and the unused residuals should not be poured back into the bottle.This product is a colorless to slightly yellow transparent solution. It should be discarded when becoming turbid or changing to a darker blue color.TMB can be irritating to the human body. Please avoid direct contact or inhalation.This product is for R&D only. Not for drug, household, or other uses.For your safety and health, please wear a lab coat and disposable gloves during the operation.
Instructions for Use： 1. For ELISA:a. Prior to use, equilibrate the TMB Chromogen Solution to room temperature.b. Following the instructions of the ELISA kit, after incubation with the HRP-labeled antibody, wash the wells 3-5 times with an appropriate wash buffer for 3-5 minutes each time. c. Remove the wash buffer and add 100μl of the TMB Chromogen Solution.d. Incubate at room temperature in the dark for 3-30 minutes, or until the desired color intensity is achieved. The chromogenic reaction is rapid and the reaction time can be shortened if necessary.e. Measure the absorbance at 370nm or 620-650nm. Alternatively, add 100μl of the Stop Solution for TMB Substrate (450nm, Sulfuric acid free) , Stop Solution for TMB Substrate (650nm, non-corrosiveness) , or 2M H2SO4 to terminate the reactions before absorbance measurement at the corresponding wavelength.2. For other assays in 96-well plates, such as detecting endogenous peroxidase in tissue or cell samples:a. Add 10-20μl of sample into each well of 96-well plates.b. Add 100μl of the TMB Chromogen Solution per well.c. Incubate at room temperature in the dark for 3-30 minutes or longer until the desired color intensity is achieved.d. Measure the absorbance at 370nm or 620-650nm. Alternatively, add 100μl of the Stop Solution for TMB Substrate (450nm, Sulfuric acid free) , Stop Solution for TMB Substrate (650nm, non-corrosiveness) , or 2M H2SO4 to terminate the reactions before absorbance measurement at the corresponding wavelength.FAQ:1. The background is too dark.a. Use an appropriate blocking buffer or serum (10%) from the same source as the primary antibody for blocking. b. Use secondary antibodies that have been appropriately adsorbed to reduce non-specific binding of secondary antibodies.c. Shorten the reaction time or reduce the concentration of secondary antibodies. d. Use an appropriate wash buffer or extend the washing time.2. No staining or weak staining.a. Increase the concentration of primary or secondary antibody. To test the effectiveness of the secondary antibody, check if the diluted secondary antibody can react with the TMB Chromogen Solution properly.b. Use a more sensitive amplification assay, such as a biotin assay.c. Extend the reaction time if necessary.d. Use a more effective primary antibody or ELISA kit.References:1. Josephy PD, Eling T, Mason RP. J Biol Chem. 1982. 257(7):3669–3675.2. Liem HH, Cardenas F, Tavassoli M, Poh-Fitzpatrick MB, Muller-Eberhard U. Anal Biochem. 1979. 98(2):388–393.
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