How does TMB work?
Background: TMB (3,3′, 5,5”-tetramethylbenzidine) is used as a substrate to generate detectable signal in ELISA. The reaction between the TMB substrate and a peroxidase, typically horseradish peroxidase (HRP), produces a measur- able color change that correlates with analyte level.
What does TMB detect?
Tetramethylbenzidine [TMB] test is a presumptive test, generally employed for the detection of blood based on reagent action of peroxide with heme to impart blue-green color.
What is TMB substrate?
TMB (3,3′,5,5′-Tetramethylbenzidine) is a substrate for horseradish peroxidase (HRP), a common label conjugated to antibodies for applications such as immunohistochemistry, western blotting, and ELISA. The combination of HRP and hydrogen peroxide causes the oxidation of TMB, resulting in the formation of a blue color.
What color is TMB substrate?
blue color
TMB (3,3′,5,5′-tetramethylbenzidine) is a chromogen that yields a blue color when oxidized, typically as a result of oxygen radicals produced by the hydrolysis of hydrogen peroxide by HRP. For kinetic or non-stopped ELISA assays, the TMB chromogen has maximal absorbances at 370nm and 652nm.
What is substrate solution?
The TMB High Sensitivity (TMB HS) Substrate solution is an one-step working solution for HRP (horse-radish peroxidase) labeled conjugates. TMB HS substrate solution provides high signal and high sensitivity for ELISA. It is recommended for cytokine ELISA reagent sets.
What is a substrate solution?
How do you make a TMB substrate?
A TMB stock solution can be prepared by dissolving 1 mg/ml in DMSO. The working solution is made by diluting 1 ml of the DMSO stock with 9 ml phosphate- citrate buffer, pH 5.0, and then adding 2 µl of fresh 30% hydrogen peroxide (Catalog Number H1009) per 10 ml of substrate/buffer solution immediately prior to use.
Does TMB contain H2O2?
Product Description: TMB No Hydrogen Peroxide One Component Substrate contains 3,3′,5,5′-tetramethylbenzidine in a mildly acidic buffer. The substrate is supplied as a ready to use solution for hydrogen peroxide assays or products producing hydrogen peroxide.
Is TMB toxic?
Harmful by inhalation, in contact with skin and if swallowed. Irritating to eyes, respiratory system and skin. Very toxic to aquatic organisms, may cause long-term adverse effects in the aquatic environment.
Is TMB substrate light sensitive?
Recommended Handling Please note the following precautions when handling neogen’s TMB substrates: Light Exposure -TMB is very light sensitive and direct exposure to sunlight should be avoided. Prolonged exposure of the substrate to light (especially sunlight) should be avoided.
Why does TMB turn yellow?
Exposing TMB to a large excess of H2O2 over TMB leads to the complete conversion of all the TMB (diamine form) into the two-electron-loss oxidation state yellow colored diimine form.
How do you prepare a TMB assay solution?
To prepare a working solution, mix equal volumes of TMB Substrate A and TMB Substrate B. Prepare only the amount needed for each assay run. After wells are completely washed, to remove unbound antibody and HRP, add 100 µl to 200 µl substrate solution to each well.
What is a TMB substrate?
TMB Substrate is the mixture of equal volumes of TMB Substrate A with TMB Substrate B. For example, for one plate, mix 5.5 ml TMB Substrate A with 5.5 ml of TMB Substrate B in a clean container.
How to prepare and stabilize TMB peroxidase substrate solution?
Mix 25 µl of TMB substrate to 1.0 ml of phosphate-citrate buffer plus perborate, stable for several hours at room temperature or for few days at 4 °C. Protocol for TMB Peroxidase Substrate Solution? I am going to design a commercial ELISA kit, I need to know how I prepare and stabilize TMB Substrate Solution?
What is the difference between TMB a and TMB B?
TMB Substrate A should be clear and colorless. TMB Substrate B should be colorless to a very light amber. TMB Substrate is the mixture of equal volumes of TMB Substrate A with TMB Substrate B. For example, for one plate, mix 5.5 ml TMB Substrate A with 5.5 ml of TMB Substrate B in a clean container.