TMB-PS (TMBZ-PS) is a chromogenic reagent for the detection of hydrogen peroxide e.g. in the enzymatic analysis of body fluids.

Instructions
I. Solution preparation
1. Stock solution: Dissolve TMB-PS in deionized water or an appropriate buffer, usually 1–10 mM stock solution.
2. Working solution: Dilute the stock solution to a working concentration, usually 0.1–1 mM, according to the experimental requirements. To enhance the reaction rate and sensitivity, it is recommended to use a solution containing hydrogen peroxide as the substrate solution.
II. Operation steps
1. Enzyme-linked immunosorbent assay (ELISA):
(1) Add specific antibodies or antigens to the wells of the ELISA plate and incubate.
(2) Add an enzyme marker that binds to the target antibody or antigen.
(3) Wash the plate with an appropriate buffer.
(4) Add TMB-PS substrate solution (usually containing hydrogen peroxide) and incubate in the dark for a certain period of time (5–30 minutes) until a blue or purple reaction product is observed.
(5) Stop reaction: Add an acidic solution (such as 2 M sulfuric acid or 1 M phosphoric acid) to terminate the reaction. The reactant changes from blue to yellow, which facilitates the reading of optical density.
2. Immunohistochemical staining:
(1) Add TMB-PS substrate solution to tissue sections or cell cultures for staining.
(2) Wash off excess dye with PBS and observe under a microscope.
3. Calibration and control:
(1) Control group: Set up a group without substrate solution as a control to ensure the specificity of the substrate reaction.
(2) Standard curve: A standard curve between optical density and concentration can be established using samples of known concentration for quantitative analysis.
Notes:
(1) Photosensitivity: TMB-PS is light-sensitive and should be avoided from prolonged exposure to strong light.
(2) Storage conditions: TMB-PS should be stored in a dry, cool place, away from high temperatures and strong light, usually at -20°C.
(3) pH range: TMB-PS performs best in an acidic environment of pH 3–5, but is also stable over a wider pH range.
(4) Solubility: TMB-PS has good solubility in water, but it should be ensured that there are no undissolved solid particles.