b'Chromogenic HRP substrates the product results in dark blue bands where the enzyme There are numerous options for chromogenic HRPis located. 1-Step Ultra TMB-Blotting Solution is well suited substrates, but all require the presence or addition offor applications that require a high signal-to-noise ratio. peroxide for colorimetric detection (Figure 35). Because ofDAB yields a brown precipitate in the presence of HRP its extremely short shelf life at the desired concentration,and peroxide. The brown, insoluble product can be readily hydrogen peroxide traditionally was added to a buffer,chelated with osmium tetroxide. The color produced by along with the substrate, immediately prior to use. As aDAB can be intensified with the addition of metals such result, these substrates typically had a useful shelf life ofas nickel, copper, silver, and cobalt that form complexes. only a few hours. Many commercially available precipitatingThe color produced by the metal complexes is darker than HRP substrates are supplied with, or come preparedthe color produced by DAB alone, enhancing the sensitivity in, stable peroxidase substrate buffer. The stabilizedin staining applications. Chloronaphthol (4-chloro-1-peroxide in these solutions is generally concentrated andnaphthol, 4-CN) can be used for chromogenic detection less corrosive than the traditional 30% stock solution ofof HRP in western blotting and immunohistochemistry. hydrogen peroxide. Since 30% hydrogen peroxide andThis precipitate is not as sensitive or as stable as TMB dilution solutions of hydrogen peroxide are not stable,and DAB, but the alcohol-soluble precipitate photographs reagents prepared with stabilized peroxide will providewell and has a distinct blue-purple color that can be useful more consistent results. TMB is most often used as ain double-staining applications. The individual benefits of substrate for HRP in ELISAs. However, in the presence4-CN and DAB are often combined into a single substrate of HRP and peroxide, a water-soluble blue product ismixture, such as the CN/DAB solution used in the Thermo generated that can be precipitated onto a membrane.Scientific CN/DAB Substrate Kit. CN/DAB substrate has Thermo Scientific 1-Step Ultra TMB-Blotting Solutionexcellent sensitivity, yielding a dark black precipitate that is a single-component peroxidase substrate for westernphotographs well. CN/DAB substrate works well in western blotting and immunohistochemistry (Table 21). Precipitatingblotting and dot blotting applications.H 3 C CH 3 OH H NNH 22H 2 N NH 2 H 2 N NH 2H 3 C CH 3 ClTMB 4-CN DABMW: 240.4 MW: 178.6 MW: 214.1Figure 35. Chromogenic substrates for western blotting with HRP.Table 21. Properties of Thermo Scientific HRP-based chromogenic substrates.Metal Enhanced DAB1-Step Ultra TMB- Pierce CN/DAB1-Step Chloronaphthol Substrate Kit Blotting Solution Substrate Kit Substrate SolutionDetection limit 17 pg 20 pg 500 pg 5 ngColor Brown-black Dark blue Black Blue-purpleFormat 2-component reagent kit Ready-to-use solution Ready-to-use solution 2-component reagent kitRecommended antibody1: 1:1,000 1: 1:1,000 1: 1:500 1: 1:500dilutions (from 1 mg/mL stock) 2: 1:5,0001:50,000 2: 1:5,0001:10,000 2: 1:2,0001:20,000 2: 1:2,0001:20,000Cat. No.34065 37574 34000 3401255'