Enhanced TMB Chromogen Solution for ELISA

Cat. No.: E743397

Overview

The Enhanced TMB Substrate Solution for ELISA adopts the latest color development technology with a single TMB solution. The conversion of TMB into a soluble blue product is catalyzed by horseradish peroxidase (HRP). This product can also be used to assay the content of peroxidase in blood or hemoglobin samples.The traditional TMB color development reagents consists of multiple component that must be prepared freshly before use and are prone to precipitation, which is relatively inconvenient and produces inconsistent assay results easily. This product is a ready-to-use single-component solution, which greatly simplifies the procedures and makes the assay more robust and reliable.TMB, namely 3, 3',5 ,5'-Tetramethylbenzidine, is a substrate of HRP. Under the catalysis of HRP or other appropriate peroxidases, TMB generates a soluble blue product with an absorbance at 370nm or 620-650nm. Please refer to Figure 1 for the assay results of HRP-labeled secondary antibodies at different concentrations using this product.Figure 1. The assay results of HRP-labeled secondary antibodies at different concentrations using Aladdin's Enchanced TMB Substrate Solution for ELISA . A. Comparison of the assay results between the TMB Substrate Solution for ELISA and the Enhanced TMB Substrate Solution for ELISA . B. Measurement of A370 before the addition of Stop Solution and A450 after the addition of Stop Solution. As shown in the figure, the concentration of HRP-labeled secondary antibody shows good linearity in the range of 0-0.02% and the assay sensitivity of P2010 is approximately 50% higher than that of P0209. The assay sensitivity is higher after the addition of stop solution . This figure is for reference only, which may vary due to different experimental conditions.After TMB conversion into the blue product by HRP, the reaction can be stopped by the addition of Stop Solution for TMB Substrate (450nm, Sulfate Free) , Stop Solution for TMB Substrate (650nm, Non-corrosive) or home-made 2M H2SO4. After adding Stop Solution for TMB Substrate (450nm, Sulfate Free) or 2M H2SO4, the solution turns yellow and has a higher absorbance value at 450nm (Figure 1B). After adding Stop Solution for TMB Substrate (650nm, Non-corrosive), the solution remains blue and the absorbance can be measured at 620-650nm (Figure 1B).This product is most commonly used in ELISA, but can also be used to determine the content of peroxidase in blood or hemoglobin samples. When used for ELISA, each sample usually requires 0.1ml of this product.

Precautions：

TMB is irritating. Please take effective measures to avoid inhalation or direct contact with skin.This product is a colorless to slightly blue transparent solution. Discard it when it turns turbid or darker blue.This product is for R&D only. Not for drug, household, or other uses.For your safety and health, please wear a lab coat and disposable gloves during the operation.

Instructions for Use：

1. For ELSIA:a. (Refer to the instructions of the ELISA kit) After incubation with HRP-labeled antibody, wash 3-5 times with an appropriate wash buffer for 3-5 minutes each.b. Remove the wash buffer and add 200μl of TMB Substrate Solution for ELISA.c. Incubate at room temperature in the dark for 3-30 minutes or longer (up to 24 hours) until the desired color intensity is achieved.d. Measure the absorbance directly at 370nm or 620-650nm. Alternatively, add 100μl of Stop Solution for TMB Substrate (450nm, Sulfate Free) (, P0215), Stop Solution for TMB Substrate (650nm, non-corrosive) (, P0217) or home-made 2M H2SO4 to terminate the reaction, then measure the absorbance.2. For other assays in 96-well plates (e.g., detection of endogenous peroxidase in cell or tissue samples):a. Add 10-20 microliters of sample directly to each well of 96-well plates.b. Add 200μl of MB Substrate Solution for ELISA.c. Incubate at room temperature in the dark for 3-30 minutes or longer (up to 24 hours) until the desired color intensity is achieved.d. Measure the absorbance directly at 370nm or 620-650nm. Alternatively, add 100μl of Stop Solution for TMB Substrate (450nm, Sulfate Free) (, P0215), Stop Solution for TMB Substrate (650nm, non-corrosive) (, P0217) or home-made 2M H2SO4 to terminate the reaction, then measure the absorbance.FAQ：1. Too dark backgrounda. If the background of negative control is too dark, attempt other appropriate blocking buffers or block with 10% serum from the same source as the primary antibody. Alternatively, use secondary antibodies that have been properly adsorbed to reduce non-specific bindings of secondary antibody.b. Reducing the color development time or the concentration of secondary antibody can be considered. Using other wash buffers with stronger strength or extending the washing time may also help.2. No color or too weak colora. Increase the concentration of the primary or secondary antibody appropriately. To test the effectiveness of the secondary antibody, add a drop of diluted secondary antibody to a centrifuge tube and check if the colour can be developed normally.b. Use a more sensitive detection system with higher amplification efficiency, such a biotin detection system.c. Extend the color development time appropriately.d. Use a primary antibody or ELISA kit with better effects.

Specifications

Product Name

Enhanced TMB Chromogen Solution for ELISA

Note

pH value: 3.6±0.4

Molecule Type

Unknown

Storage and Shipping

Storage

Store at 2-8°C,Protected from light

Shipped In

Wet ice

Stability And Storage

Store at 4℃ in the dark for up to 1 year.

Precautions：

TMB is irritating. Please take effective measures to avoid inhalation or direct contact with skin.