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.
