| Size | Price | Stock | Qty |
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| 1g |
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| Other Sizes |
| Targets |
The primary target of TMB is horseradish peroxidase (HRP, EC 1.11.1.7), a heme-containing enzyme commonly used as a label in immunoassays. In the presence of hydrogen peroxide (H2O2), HRP catalyzes the one-electron oxidation of TMB to form a blue charge-transfer complex (TMB radical cation), which can be further oxidized to a yellow diimine product after acidification (H2SO4 or H3PO4) for measurement at 450 nm. TMB also detects reactive oxygen/nitrogen species (ROS/RNS).
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| ln Vitro |
TMB (dihydrochloride x.hydrate) can bind tightly to Escherichia coli ATCC 25922 cells instead of being internalized, thereby detecting singlet oxygen produced on the outer membrane [1].
In vitro, TMB (0.1-1 mg/mL) is used in HRP-based assays. In a typical ELISA, HRP-labeled detection antibody oxidizes TMB in the presence of 0.01-0.03% H2O2, producing a blue color (A₆₅2). After stopping with 2 M H2SO4, the yellow product is read at A4₅0. The detection limit for HRP is as low as 10-¹⁷ mol. TMB is superior to other substrates (e.g., ABTS, OPD) due to low background, high sensitivity, and lack of carcinogenicity. It also reacts with hypochlorite, singlet oxygen, and NO2 to produce blue oxidation products. In bacterial studies, TMB detects singlet oxygen produced by E. coli. |
| ln Vivo |
In vivo, TMB dihydrochloride is not typically used; it is an in vitro diagnostic reagent. However, it has been explored as a photoacoustic probe for ROS/RNS detection in animal tissues. Localized injection into tumor tissues followed by photoacoustic imaging can map ROS production. No significant in vivo activity beyond diagnostic applications.
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| Enzyme Assay |
For HRP enzymatic assays, 96-well microplates are coated with capture antibody and incubated with antigen and HRP-labeled detection antibody. TMB solution: TMB dihydrochloride (0.1 mg/mL) is dissolved in 50 mM phosphate-citrate buffer (pH 5.0) containing 0.01-0.03% H2O2. Add 100 uL/well, incubate 5-30 min at RT. The reaction is stopped by adding 100 uL of 2 M H2SO4 or 0.5 M H3PO4. Absorbance is read at 450 nm (for yellow product) or 652 nm (for blue product). For kinetic assays, A₆₅2 is measured continuously every 30 sec for 10 min. For singlet oxygen detection, a suspension of E. coli ATCC 25922 cells is incubated with TMB (10-50 uM) for 30 min at 37degC. Blue color development (A₆₅2) is measured as an indicator of singlet oxygen production.
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| Cell Assay |
For cell-based assays (e.g., cell ELISA, ELISpot), cells are fixed and permeabilized, blocked, and incubated with HRP-labeled antibodies. TMB substrate is added, and the blue color is observed microscopically or quantified in solution after cell lysis. For ROS detection in cells, cells are incubated with TMB (10-100 uM) in PBS for 10-30 min after stimulation (e.g., LPS, H2O2), and the blue oxidation product is measured in cell lysates by A₆₅2. For cytotoxicity, TMB is non-toxic; no effect on viability up to 1 mM.
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| Animal Protocol |
For in vivo ROS/RNS detection, TMB (10-100 uL of 1-10 mg/mL in PBS) is injected intratumorally or intravenously into tumor-bearing mice. After 10-30 min, the animal is subjected to photoacoustic imaging (Excitation 680-750 nm). Signal intensity correlates with ROS/RNS levels in the tissue. For diagnostic purposes, animals are not used routinely. Not applicable for efficacy.
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| ADME/Pharmacokinetics |
The dihydrochloride salt is highly water-soluble (soluble in water to at least 1 mg/mL with heating to 45degC and sonication). Stock solutions (0.1 mg/mL in 50% acetic acid or phosphate-citrate buffer) are stable for months at 4degC when tightly sealed (protect from light). The working solution is prepared fresh immediately before use. In vivo, TMB is rapidly cleared; not suitable for systemic administration.
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| Toxicity/Toxicokinetics |
TMB dihydrochloride is a non-toxic, non-mutagenic chromogen. It is not a hazardous substance. However, the powder may cause mild skin/eye irritation; use PPE. No carcinogenicity. Safe for routine laboratory use.
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| References |
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| Additional Infomation |
TMB is the most widely used HRP substrate for ELISA, Western blotting, and IHC. It is an in vitro diagnostic reagent, not an approved drug. Developed in 1979, it has supplanted carcinogenic substrates such as OPD and ABTS. For research use only. Not for human therapeutic use.
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| Molecular Formula |
C16H22CL2N2.XH2O
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|---|---|
| Molecular Weight |
313.2653
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| Exact Mass |
330.127
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| CAS # |
207738-08-7
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| PubChem CID |
19083738
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| Appearance |
Light brown to brown solid powder
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| Boiling Point |
459.8ºC at 760 mmHg
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| Melting Point |
>300ºC
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| Flash Point |
231.9ºC
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| Vapour Pressure |
3.01E-09mmHg at 25°C
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| LogP |
6.518
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| Hydrogen Bond Donor Count |
5
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| Rotatable Bond Count |
1
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| Heavy Atom Count |
21
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| Complexity |
226
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| Defined Atom Stereocenter Count |
0
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| SMILES |
Cl[H].Cl[H].N([H])([H])C1=C(C([H])([H])[H])C([H])=C(C([H])=C1C([H])([H])[H])C1C([H])=C(C([H])([H])[H])C(=C(C([H])([H])[H])C=1[H])N([H])[H]
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| InChi Key |
KVCWTKDFVVSVSJ-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C16H20N2.2ClH.H2O/c1-9-5-13(6-10(2)15(9)17)14-7-11(3)16(18)12(4)8-14;;;/h5-8H,17-18H2,1-4H3;2*1H;1H2
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| Chemical Name |
4-(4-amino-3,5-dimethylphenyl)-2,6-dimethylaniline;hydrate;dihydrochloride
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| Synonyms |
Sure Blue TMB dihydrochloride x.hydrate
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples
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| Solubility (In Vivo) |
Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution. Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil) Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals). View More
Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)] Oral Formulations
Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium) Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 3.1921 mL | 15.9607 mL | 31.9213 mL | |
| 5 mM | 0.6384 mL | 3.1921 mL | 6.3843 mL | |
| 10 mM | 0.3192 mL | 1.5961 mL | 3.1921 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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