TMB monosulfate - BioReagent , CAS No.54827-18-8

CAS: 54827-18-8 Cat. No.: T303960 Fórmula: C₁₆H₂₂N₂O₄S Peso molecular: 338.42 PubChem CID: 67612326

GRADE & PURITY BioReagent

Synonyms

TMB monosulfate | Sure Blue TMB monosulfate | TMB (monosulfate) | 4-(4-amino-3,5-dimethylphenyl)-2,6-dimethylaniline | 3,3',5,5'-Tetramethyl-[1,1'-biphenyl]-4,4'-diamine sulfate | 3,3',5,5'-tetramethylbenzidine sulfate | BM blue monosulfate | SCHEMBL85210

Storage

Store at -20°C

Shipped In

Ice chest + Ice pads

Visão geral

TMB monosulfate is a chromogenic substrate used in staining procedures in immunohistochemistry as well as being a visualizing reagent used in enzyme-linked immunosorbent assays (ELISA).

Specifications

Sinónimos

TMB monosulfate | Sure Blue TMB monosulfate | TMB (monosulfate) | 4-(4-amino-3,5-dimethylphenyl)-2,6-dimethylaniline | 3,3',5,5'-Tetramethyl-[1,1'-biphenyl]-4,4'-diamine sulfate | 3,3',5,5'-tetramethylbenzidine sulfate | BM blue monosulfate | SCHEMBL85210

Especificações e pureza

BioReagent

Condições de armazenamento de armazenamento

Store at -20°C

Enviado em

Ice chest + Ice pads

Este produto requer transporte de cadeia fria. Serviços terrestres e outros serviços econômicos não estão disponíveis.

Grau

BioReagent

Nomes e identificadores

Pubchem Sid

504771906

Pubchem Sid Url

https://pubchem.ncbi.nlm.nih.gov/substance/504771906

Sorrisos canónicos

CC1=CC(=CC(=C1N)C)C2=CC(=C(C(=C2)C)N)C.OS(=O)(=O)O

IUPAC Name

4-(4-amino-3,5-dimethylphenyl)-2,6-dimethylaniline;sulfuric acid

InChIKey

VJSXOBXXVSDFKR-UHFFFAOYSA-N

INCHI

1S/C16H20N2.H2O4S/c1-9-5-13(6-10(2)15(9)17)14-7-11(3)16(18)12(4)8-14;1-5(2,3)4/h5-8H,17-18H2,1-4H3;(H2,1,2,3,4)

SMILES isoméricas

CC1=CC(=CC(=C1N)C)C2=CC(=C(C(=C2)C)N)C.OS(=O)(=O)O

PubChem CID

67612326

Peso molecular

338.42

Propriedades químicas e físicas

• Peso molecular: 338.400 g/mol
• Hydrogen Bond Donor Count: 4
• Hydrogen Bond Acceptor Count: 6
• Rotatable Bond Count: 1
• Exact Mass: 338.13 Da
• Monoisotopic Mass: 338.13 Da
• Topological Polar Surface Area: 135.000 Ų
• Heavy Atom Count: 23
• Formal Charge: 0
• Complexity: 308.000
• Isotope Atom Count: 0
• Defined Atom Stereocenter Count: 0
• Undefined Atom Stereocenter Count: 0
• Defined Bond Stereocenter Count: 0
• Undefined Bond Stereocenter Count: 0
• The total count of all stereochemical bonds: 0
• Covalently-Bonded Unit Count: 2

XLogP3

Application Protocols

No item-specific tested applications or protocols are provided in the Product Data for SKU T303960. Refer to the product datasheet and your assay SOPs for validated procedures.

General literature example (for context only; not a product specification):

• Equilibrate plate and reagents to room temperature. Add TMB monosulfate substrate to wells containing HRP conjugate. Develop color for a defined time while protected from light. For endpoint assays, add acid stop and read at 450 nm (reference 620–650 nm if available). Optimize incubation time and substrate concentration for your specific assay to balance sensitivity and background.

Biological Roles

This product is a synthetic chromogenic substrate and does not have a native biological role in cells or organisms.

Relevant literature context:

• Serves as an electron donor for peroxidase enzymes in analytical assays; not a metabolite or signaling molecule.
• The HRP–TMB–H2O2 system is widely used to transduce biomolecular recognition events (e.g., antigen–antibody binding) into measurable optical signals, but the substrate itself is not biologically active under physiological conditions.

For pathway or enzymology discussions, refer to peroxidase catalytic cycles and one-electron oxidation chemistry rather than metabolic pathways.

Buffer Applications

As a chromogenic substrate, TMB monosulfate is not a buffering agent. However, buffer choice strongly influences its assay performance.

Literature-based guidance:

• Preferred pH range: mildly acidic (approximately pH 3.5–6.0) supports substrate stability and robust HRP activity; optimal pH depends on the assay format.
• Common buffers: acetate (pH ~4–6), citrate–phosphate (pH ~4–6), MES (pH ~5.5–6.5). Use metal-free reagents and deionized water to minimize auto-oxidation.
• Ionic strength: moderate ionic strength helps maintain reproducibility while avoiding enzyme inhibition.
• Stop solution: strong acid (e.g., sulfuric acid) is typically used to convert the blue intermediate to the yellow diimine measured at 450 nm for endpoint ELISA.

Note: Exact buffer composition and concentration should be validated per assay. No item-specific buffer recipes are provided for this product.

Green Alternatives

Context

• Chromogenic HRP substrates differ in hazard profile, waste classification, and environmental persistence. Selection can reduce solvent use and hazardous waste.

Comparative overview (literature; general guidance)

• TMB (monosulfate) vs OPD (o-phenylenediamine): TMB offers higher sensitivity and less mutagenic concern than OPD, but still requires careful handling and waste management.
• TMB vs ABTS: ABTS is highly water-soluble and yields a green product measurable at ~405 nm; TMB often provides greater sensitivity and lower background in ELISA but can be more light-sensitive.
• TMB vs AEC/DAB: AEC and DAB are used for insoluble end-products in histochemistry; DAB is considered more hazardous. TMB in solution-phase ELISA can be a greener choice by avoiding precipitating chromogens.

Operational greening tips

• Use aqueous buffers exclusively; avoid organic cosolvents.
• Prepare only the amount of substrate needed to minimize disposal.
• Neutralize and collect colored waste for appropriate hazardous disposal; avoid drain disposal.
• Consider microplate miniaturization to reduce reagent volumes and waste generation.

Pharmaceutical Uses

No pharmaceutical or excipient uses are specified for this item. TMB monosulfate is supplied for research use only and is typically employed as a chromogenic substrate in laboratory assays (e.g., ELISA) rather than in drug product formulations.

If a regulated diagnostic application is intended, additional qualification and validation beyond BioReagent grade would be required; consult applicable regulatory standards. This listing does not include such qualifications.

Physical Properties

Item-specific data

• Appearance: Not specified for this item; refer to CoA/Spec Sheet.
• Molecular weight: Not specified for this item; refer to CoA/Spec Sheet.

Storage and shipping (from Product Data)

• Storage: Store at −20 °C
• Shipped: Ice chest + ice pads

Literature/typical properties (for context; not item specifications)

• Physical form: commonly supplied as a solid sulfate salt that is more water-compatible than free-base TMB.
• Solubility (literature): TMB monosulfate is designed for ready dissolution in water or mildly acidic buffers; the free base is sparingly soluble in water but soluble in organic solvents. The sulfate salt increases aqueous solubility markedly.
• Spectral behavior (literature, HRP assays):
  • Reduced TMB is essentially colorless.
  • HRP/H2O2 oxidation yields a blue cation-radical product with Amax ~650 nm and a secondary band ~370 nm.
  • Upon acid stop (e.g., sulfuric acid), the product converts to a yellow diimine with Amax ~450 nm.

Note: Where exact numeric values (mp, density, logP, pKa, refractive index) are needed for method validation, consult the item-specific CoA/SDS; such data are not specified for this item.

Quality and Grades

Item-specific grade

• Grade: BioReagent (from Product Data)

What BioReagent typically implies (general guidance)

• Intended for biochemical assays and general laboratory research. Emphasis on purity suitable for enzymatic colorimetric assays (e.g., peroxidase-based ELISAs) and minimized assay-interfering impurities.
• Not intended for clinical/diagnostic use without further qualification.

Stabilizer and counterion considerations (general)

• The sulfate salt form of TMB is commonly selected to improve aqueous solubility and solution stability relative to the free base, reducing spontaneous oxidation background. If stabilizers are present, they will be listed on the CoA/SDS; none are specified for this item.

Verification

• For exact acceptance criteria (assay, maximum impurities, water, inorganic residue, UV profile), and for any stabilizers, refer to the item’s CoA/Spec Sheet. No additional item-specific specifications are provided here.

Reaction and Applications

Primary application (literature)

• Substrate for peroxidase enzymes (notably horseradish peroxidase, HRP). In the presence of H2O2, HRP catalyzes one-electron oxidation of TMB to a blue charge-transfer/cation-radical species (Amax ~650 nm); acidification converts it to a stable yellow diimine (Amax ~450 nm) suitable for endpoint measurement.

Use cases

• ELISA and immunoassays as a chromogenic reporter for HRP-conjugated detection reagents.
• Blot/spot assays, peroxidase activity assays, and microplate-based kinetic studies.

Mechanistic notes (literature)

• HRP Compound I abstracts an electron from TMB, generating a radical cation; sequential oxidation and dimer interactions produce the characteristic blue species. Proton activity and buffer composition modulate the relative distribution of blue intermediate and yellow diimine.

Practical tips

• Prepare substrate solutions immediately before use; protect from light.
• Use metal-free plasticware or chelators (if validated) to limit non-enzymatic oxidation.
• Control H2O2 concentration carefully; excess peroxide accelerates background and may inactivate HRP.
• For endpoint assays, add stop acid uniformly and read promptly at 450 nm with reference 620–650 nm if your instrument supports it.

Note: The above are literature-based good practices; this product’s Manufacturer Applications field does not add item-specific instructions.

Reaction Conditions

The following conditions are general literature guidance for HRP–TMB assays and are not item-specific specifications.

• Solvent/buffer: acetate or citrate–phosphate buffer, pH ~4.0–6.0; protect solutions from light.
• Temperature: ambient (20–25 °C) for most ELISA applications; maintain consistent temperature across plates for reproducibility.
• Substrate preparation: dissolve TMB monosulfate in buffer to the assay’s recommended concentration immediately before use. Filter if necessary through 0.22 µm to reduce particulates.
• Oxidant: H2O2 at low millimolar to micromolar range depending on sensitivity requirements; prepare fresh to limit decomposition.
• Reaction time: typically 5–30 minutes for ELISA development; monitor color formation kinetically (650 nm) or proceed to acid stop and read at 450 nm.
• Quench: add sulfuric acid (concentration per protocol) to convert to the yellow end-product for endpoint readings.
• Expected performance: linear dynamic ranges and limits of detection depend on enzyme conjugate loading, plate type, and background; optimize empirically.

Cautions:

• Excess peroxide can increase background and reduce HRP activity.
• Transition metal contamination and alkaline pH promote non-enzymatic oxidation; use clean, metal-free ware and validated buffers.

Safety and Handling

GHS and hazard information (item-specific)

• Signal word: Not specified for this item; refer to SDS.
• Hazard statements (H-phrases): Not specified for this item; refer to SDS.
• GHS classification/pictograms: Not specified for this item; refer to SDS.

General safety guidance (literature/practice; not a substitute for SDS)

• TMB derivatives are aromatic diamines; handle with care to avoid skin contact, inhalation, or ingestion. Some benzidine-class compounds are suspected mutagens; minimize exposure.
• PPE: lab coat, safety glasses, and appropriate chemically resistant gloves (e.g., nitrile). Work in a fume hood when preparing powders/solutions and when handling oxidants (H2O2).
• Incompatibilities: strong oxidizers will rapidly convert reduced TMB to colored species; keep substrate solutions protected from light and away from metal contaminants. Avoid strong bases that can accelerate non-enzymatic oxidation.
• First aid (overview):
  • Skin/eye contact: rinse with water for ≥15 minutes; remove contaminated clothing; seek medical attention as needed.
  • Inhalation: move to fresh air; seek medical attention if symptoms persist.
  • Ingestion: rinse mouth; do not induce vomiting; seek medical attention.
• Waste: chromogenic oxidation products can be hazardous; collect liquid waste containing TMB oxidation products and peroxide for appropriate disposal per institutional and regulatory guidelines.
• Light sensitivity: store solutions protected from light to reduce background oxidation.

Solvent Selection

Applicability to this compound type

• TMB monosulfate is primarily used as an aqueous chromogenic substrate; solvent choice centers on buffer systems rather than organic media.

General solvent/buffer behavior (literature)

• Polarity/miscibility: as a protonated diamine sulfate salt, it dissolves readily in water and mildly acidic buffers; miscible with typical assay buffers. Organic solvents are generally unnecessary and may destabilize the reduced form.
• Preferred media: acetate, citrate–phosphate, or MES buffers at mildly acidic pH to balance enzyme activity of HRP and substrate stability.
• Avoid: strong alkaline buffers (promote auto-oxidation), metal-contaminated solutions, and exposure to light.

When to choose alternative media

• For peroxidase kinetic work requiring maximal sensitivity, select low-ionic-strength, metal-free buffers and freshly prepared H2O2.
• For endpoint ELISA with acid stop, ensure compatibility between the working solvent (buffer) and the stop solution (e.g., sulfuric acid) to avoid precipitation or spectral interferences.

Note: Specific solubility limits and preparation concentrations are method-dependent; consult your assay protocol and the CoA for item-specific recommendations.

Storage and Reconstitution

Item-specific

• Storage: Store at −20 °C (from Product Data)
• Shipping: Ice chest + ice pads (from Product Data)

General handling guidance (not item-specific specs)

• Keep container tightly closed, dry, and protected from light. Allow to equilibrate to room temperature in a desiccator before opening to minimize moisture condensation.
• Reconstitution: prepare fresh aqueous/buffered working solutions immediately prior to use. If stock solutions are prepared, aliquot into light-resistant containers, purge headspace with inert gas if possible, and freeze to minimize oxidation.
• Freeze–thaw: avoid repeated freeze–thaw of aqueous stocks; aliquot appropriately.
• Shelf life: consult the CoA/Spec Sheet for lot-specific retest or expiration dating; not specified for this item.

Always defer to the SDS and CoA for definitive storage stability, permissible concentrations, and any stabilizers or preservatives associated with this specific item.

Structure and Identity

Item-specific identifiers

• CAS: 54827-18-8 (from Product Data)
• SKU: T303960
• Grade: BioReagent
• InChIKey: Not specified for this item; refer to CoA/Spec Sheet. (Product Data entry is incomplete)
• SMILES: Not specified for this item; refer to CoA/Spec Sheet.
• Molecular formula: Not specified for this item; refer to CoA/Spec Sheet.
• Molecular weight: Not specified for this item; refer to CoA/Spec Sheet.

General structural description (literature)

• TMB monosulfate is the sulfate salt of 3,3′,5,5′-tetramethylbenzidine (TMB), a benzidine-type diamine bearing four methyl substituents (two on each anisidyl ring). In the sulfate form, the diamine is protonated and counterbalanced by sulfate, improving aqueous stability and handling.
• Key functional groups: two aniline-type amino groups (protonated in the salt), four methyl substituents on the aromatic rings, and a sulfate counterion (not covalently bound to the aromatic framework).
• 2D structure summary: two para-phenyl rings linked via a –NH–NH– bridge (benzidine core), each ring bearing ortho/para methyl substitution; protonation sites at the two amino nitrogens; sulfate as inorganic counterion.

Synthetic Utility

TMB monosulfate is principally an analytical reagent rather than a synthetic building block. Nonetheless, the underlying chemistry is informative:

• Functional groups: benzidine core with two aniline-type amino groups (protonated in the sulfate salt); readily undergoes one-electron oxidation to persistent colored species.
• Redox transformations: serves as a calibrated redox probe for peroxidase-catalyzed oxidations; the well-defined spectral shifts facilitate kinetic analyses of peroxidase mechanisms.
• Indicator utility: can function as a color indicator for oxidizing conditions and peroxidase-like nanozyme catalysis in method development.

For preparative organic synthesis, TMB is rarely used as a substrate or intermediate; its value lies in assay development, method validation, and mechanistic studies of oxidative catalysis.

Target Specificity

Not applicable. This product is a small-molecule chromogenic substrate and does not possess target specificity in the sense of antibodies or biological ligands. It is oxidized by peroxidase enzymes (e.g., HRP) in a generally non-specific manner toward the enzyme’s active site chemistry.

Perguntas frequentes

What does BioReagent mean?

BioReagent indicates that the product has been processed and tested for biological and biochemical applications. Specifications may include endotoxin limits, microbial controls, sterility, low DNase/RNase/protease activity, and verified biological activity where relevant.

How should this product be stored?

Store at ?20 °C. Freezer storage is required to maintain the specified shelf life.

How is this product shipped?

This product ships in an insulated container with ice pads. Unpack on arrival and transfer it to the storage condition stated above.

What are the CAS number, molecular formula and molecular weight?

The CAS Number is 54827-18-8, the molecular formula is C16H22N2O4S, and the molecular weight is 338.42 g/mol. InChIKey VJSXOBXXVSDFKR-UHFFFAOYSA-N.

What documentation is provided?

Available product documentation, including Certificates of Analysis (COA), Safety Data Sheets (SDS), and specification sheets, is shown in the product document area. Document availability and access follow the current site policy.