TMB 1-Component HRP Membrane Substrate (SUBM)

1. Allow product to equilibrate to room temperature (25°C) prior to use., 2. Completely cover the membrane surface with the substrate. The substrate will react with phosphatase creating a purple-blue reaction product., 3. To stop the reaction, rinse the developed membrane with distilled water., 4. For best results, the reaction should be monitored and read before the background staining becomes too intense to distinguish from the positive staining.

TMB 1-Component HRP Membrane Substrate (SUBM) is suitable for membrane applications using horseradish peroxidase (HRP) as the conjugated detection enzyme. SUBM should not be used for microWell (ELISA) applications. TMB 1 Component HRP Membrane Substrate is a one-component formulation containing stabilized 3,3’,5,5’-tetramethylbenzidine (TMB). The TMB substrate is oxidized by the peroxidase enzyme to yield an insoluble dark blue reaction product. SUBM is supplied ready-to-use at 1X. Best results are obtained by equilibrating TMB 1-Component HRP Membrane Substrate to room temperature (25°C) prior to use. After probing with the antibody and HRP reagents, wash membrane thoroughly and transfer the membrane into a clean container. Cover the membrane surface with ample amount of SUBM and incubate 5-20 minutes. The substrate will react with sites on the membrane containing peroxidase, producing an insoluble permanent dark blue reaction product. For best results, monitor the substrate color development process until the target protein bands are visible. To stop the reaction, rinse the membrane with reagent quality water. If the reaction proceeds too long, there will be excessive background staining and diminished resolution of the target peptide or protein banding regions. If the color development is too rapid or intense, it is recommended to dilute the antibodies or conjugates or shorten the incubation period.