HRP Substrate TMB Solution

Introduction

TMB solutions are chromogenic reagents for peroxidase, used for ELISA techniques. They are composed of 3,3',5,5'-tetramethylbenzidine (TMB), , and proprietary catalyzing and stabilizing agents. It produces a blue product upon interaction with HRP or HRP conjugatese . The soluble blue product can be quantitated at 650 nm. Sensitivity is greater than classic substrates like OPD and ABTS, with very low background.

Storage

2-8°C avoid light for three years. Do not freeze (Return TMB Solution to room temperature before use).

Seize:  12mL, 50 mL, 1000mL.

ELISA Applications                                                                  

1.  Wash the microplate following the incubation of HRP-labeled solution.

2.  Add 100 µL TMB Solution into each well. Incubate the plate at 37°C for 15–30 min or until the desired

     color develops.

3.  Measure the absorbance signal at 650 nm with an ELISA microplate reader.

   Note: If desired, the reaction can be stopped by adding an equal volume of 2M SC to each well. Stopped                        reaction should be read at 450 nm.

NOTE:

1.        TMB substrates are optimized for direct and indirect ELISA techniques. They are not suitable for                             Immunohistochemistry or Western Blotting.

2.        Recommendations for use: TMB is sensitive to certain conditions of storage and operating.

3.        Exposure to light : TMB substrate is light sensitive. It should be stored in amber vials of HDPE type.

4.        Material : the substrate is very sensitive to metallic ions. Only high quality plastic or glass should be used.

5.        Avoid the use of caps with rubber joins : this should impair the results.

6.        Don't pipet the TMB directly from the bottles : fill a suitable container first with the necessary solution                   volume to avoid contamination.

7.        Don't leave the bottle open too long , and keep it a dark at 2-8°C. Avoid important or frequent variations             of temp.