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613544 TMB, Soluble

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

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

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613544
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Overview

Replacement Information

Products

Catalog NumberPackaging Qty/Pack
613544-100ML Glass bottle 100 ml
Description
OverviewPeroxidase 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 Number613544
Brand Family Calbiochem®
Synonyms3,3ʹ,5,5ʹ-Tetramethylbenzidine
References
Product Information
CAS number54827-17-7
FormLiquid, clear to very light blue
FormulationSingle component system containing 1.46 mM TMB, 2.21 mM H₂O₂ in a proprietary solvent, pH 3.1 ± 0.5.
PreservativeNone
Quality LevelMQ100
Applications
Application CommentsProduct 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
RTECSDV2300000
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 InstructionsProtect 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
Catalog Number GTIN
613544-100ML 04055977263794

Documentation

TMB, Soluble SDS

Title

Safety Data Sheet (SDS) 

TMB, Soluble Certificates of Analysis

TitleLot Number
613544
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.

Revision02-September-2011 JSW
Synonyms3,3ʹ,5,5ʹ-Tetramethylbenzidine
DescriptionSoluble TMB substrate supplied as a single component system.
BackgroundTMB 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.
FormLiquid, clear to very light blue
FormulationSingle component system containing 1.46 mM TMB, 2.21 mM H₂O₂ in a proprietary solvent, pH 3.1 ± 0.5.
CAS number54827-17-7
RTECSDV2300000
PreservativeNone
CommentsProduct 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 InstructionsProtect 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