TMB Substrate Chromogenic Kit (ELISA, HRP Chromogenic)

Cat. No.: T1509497

GRADE & PURITY BioReagent

Overview

  3,3',5,5'-Tetramethylbenzidine (TMB) is an excellent chromogenic reagent for enzyme immunoassays. It is soluble in various organic solvents and double-distilled water, presenting as a stable colorless solution. When mixed with an appropriate amount of urea peroxide or hydrogen peroxide and buffer solution, it reacts with peroxidase to produce a distinct blue product that is extremely easy to observe. Due to its high sensitivity, stable working solution after preparation, and non-carcinogenic properties, TMB is widely used in enzyme-linked immunosorbent assay (ELISA) and urine test strip detection reactions, etc. TMB is mainly applied in ELISA; the classic method involves separate preparation of Solution A and Solution B, which are mixed before use and remain stable for long-term storage. This reagent is generally colorless, and sometimes slightly light blue or pale yellow.

  TMB Substrate Chromogenic Kit (for ELISA, HRP Chromogenesis) is mainly composed of TMB Chromogenic Solution, TMB Buffer, and TMB Oxidant, with the following characteristics: 1. High sensitivity; 2. Good stability with stable reading after chromogenic termination; 3. Low background: the OD value of the substrate solution detected at 450 nm is generally less than 0.05. This reagent is for research use only (RUO) and not intended for clinical diagnosis or other applications.

Materials to Be Prepared by the User

1. Microplate reader, microtiter plate or 96-well plate, incubator (optional)

2. Stop solution (optional): Hydrochloric acid, sulfuric acid, etc.

Operating Procedures (For Reference Only)

1. Take Reagent A, B and C in a suitable clean container (rinsed repeatedly with pure water), and mix them at a volume ratio of A: B: C = 5000: 5000: 4 to prepare the TMB chromogenic working solution, which can be used after reaching room temperature.

2. Solution addition: After adding HRP conjugate and incubating for a certain period, wash the plate 3-5 times, add 100 μl of TMB chromogenic working solution to each well, and incubate at room temperature (15-25℃) or 37℃ for 10-30 minutes.

3. Termination (optional): Add an equal volume of 1 M hydrochloric acid or sulfuric acid solution to terminate the reaction; the reaction solution in the wells will change from blue to yellow.

4. Reading: Measure the absorbance at 450 nm within 30 minutes.

Precautions

1. The amount of TMB Oxidant provided in this kit exceeds the actual usage; please use it in strict accordance with the specified ratio.

2. TMB Oxidant is volatile; please store it in a sealed container to avoid activity loss, and use it as soon as possible after opening.

3. TMB Oxidant is corrosive; do not allow direct contact with human skin, hair, etc.

4. High reaction background or precipitation indicates an excessively intense TMB substrate reaction.

5. Reaction termination is not a mandatory step; if no stop solution is added, perform the detection immediately after the completion of chromogenesis.

6. Commercially available concentrated hydrochloric acid is generally 11.6 M and concentrated sulfuric acid is 18.3 M; attention should be paid to their volatility.

7. To avoid precipitation, perform the reading immediately after termination, or further dilute the primary antibody and/or HRP conjugate and repeat the experiment.

8. For your safety and health, wear a lab coat and disposable gloves during operation.

9. Use the reagents as soon as possible after opening to prevent adverse effects on subsequent experimental results.

Storage and Shipping

Storage

Store at 2-8°C,Protected from light

Shipped In

Wet ice

Stability And Storage

Each component has a shelf life of 1 year under corresponding storage conditions.

Contents & Storage

T1509497	Component	2×50 mL	2×100 mL	Storage
T1509497A	TMB Chromogenic Solution	50 mL	100 mL	2-8℃. Store in the dark.
T1509497B	TMB Buffer	50 mL	100 mL	2-8℃.
T1509497C	TMB Oxidant	100 μl	2×100 μl	2-8℃. Store in the dark.