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613544 Sigma-AldrichTMB, Soluble

TMB, Soluble MSDS (material safety data sheet) or SDS, CoA and CoQ, dossiers, brochures and other available documents.

SDS
CoA
Data Sheet

Synonyms: 3,3ʹ,5,5ʹ-Tetramethylbenzidine

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Overview

Replacement Information

Pricing & Availability

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Description
Overview	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.
Catalogue Number	613544
Brand Family	Calbiochem®
Synonyms	3,3ʹ,5,5ʹ-Tetramethylbenzidine

References

Product Information
CAS number	54827-17-7
Form	Liquid, clear to very light blue
Formulation	Single component system containing 1.46 mM TMB, 2.21 mM H₂O₂ in a proprietary solvent, pH 3.1 ± 0.5.
Preservative	None
Quality Level	MQ100

Applications
Application Comments	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.

Applications

Application Comments

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.

Biological Information

Physicochemical Information

Dimensions

Materials Information

Toxicological Information

Safety Information according to GHS
RTECS	DV2300000

Safety Information

Product Usage Statements

Storage and Shipping Information
Ship Code	Ambient Temperature Only
Toxicity	Irritant
Storage	+2°C to +8°C
Protect from Light	Protect from light
Do not freeze	Ok to freeze
Special Instructions	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.

Packaging Information

Transport Information

Supplemental Information

Specifications

Global Trade Item Number
Catalogue Number	GTIN
613544-100ML	04055977263794

Documentation

TMB, Soluble SDS

Title
Safety Data Sheet (SDS)

TMB, Soluble Certificates of Analysis

Title	Lot Number
613544	Enter Lot Number

Data Sheet

Note that this data sheet is not lot-specific and is representative of the current specifications for this product. Please consult the vial label and the certificate of analysis for information on specific lots. Also note that shipping conditions may differ from storage conditions.

Revision	02-September-2011 JSW
Synonyms	3,3ʹ,5,5ʹ-Tetramethylbenzidine
Description	Soluble TMB substrate supplied as a single component system.
Background	TMB is the most sensitive chromogenic substrate for detection of horseradish peroxidase (HRP)-labeled probes. In the presence of HRP and hydrogen peroxide (H₂O₂), TMB is oxidized first to a blue, cation free radical having an absorption maximum at 653 nm (ε = 3.9 x 10⁴ M^-1cm^-1). Upon further reaction with HRP/H₂O₂, or addition of acid, the radical is converted to a terminal oxidation product, a yellow diimine that absorbs light at 450 nm (ε = 5.9 x 10⁴ M^-1cm^-1). Increased sensitivity (2-4 fold) is afforded by the yellow diimine, as its molar extinction coefficient is greater than that of the blue radical. This TMB substrate is a safe, single-component system for use in HRP-based ELISA. It is optimized with respect to TMB and H₂O₂ concentrations and yields a linear response with the concentrations of HRP usually employed in immunologic assays. Upon reaction with HRP and H₂O₂, the reagent yields a blue soluble end product that is measured at 650 nm. The color formation as a function of time can be recorded or the reaction can be stopped with sodium fluoride for endpoint determinations. Increased sensitivity can be achieved by stopping the reaction with acid, which converts the blue radical to the yellow diimine that is measured at 450 nm.
Form	Liquid, clear to very light blue
Formulation	Single component system containing 1.46 mM TMB, 2.21 mM H₂O₂ in a proprietary solvent, pH 3.1 ± 0.5.
CAS number	54827-17-7
RTECS	DV2300000
Preservative	None
Comments	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.
Storage	Protect from light +2°C to +8°C
Do Not Freeze	Ok to freeze
Special Instructions	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.
Toxicity	Irritant

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