b'Chromogenic AP substratesNitro blue tetrazolium (NBT) is a member of a class ofThis reaction proceeds at a steady rate, allowing accurate heterocyclic organic compounds known as tetrazoliumcontrol of its relative sensitivity. NBT/BCIP characteristically salts. Upon reduction, the compound yields NBT-formazan,produces sharp band resolution with little background a highly colored, water-insoluble product. The substrate isstaining of the membrane.widely used for immunochemical assays and techniques because the color produced by the formazan is linear andTable 22. Properties of Thermo Scientific AP-based stable over a wide dynamic range. 5-bromo-4-chloro- chromogenic substrate.3-indolyl phosphate (BCIP) hydrolysis by AP results1-Step NBT/BCIP in a blue-purple precipitate that can be deposited onSubstrate Solutionnitrocellulose or nylon membranes. BCIP can be used asDetection limit 30 pga chromogenic substrate for both immunoblotting andColor Black-purpleimmunohistochemical studies. An ideal system for blottingFormat Ready-to-use solutionapplications with AP is the combination of NBT and BCIP. Together, they yield an intense black purple precipitateRecommended antibody dilutions1: 1:500(from 1 mg/mL stock) 2: 1:5,0001:50,000that provides much greater sensitivity than either substrate alone (Table 22 and Figure 36). Cat. No.34042Cl O Cl Cl Cl OO ClBr Br Br Br BrO POH AP OH Tautomerism O -2HN OH N +HPO 3N N NH H H NBT NBT-formazan H HBCIP 5,5-dibromo-4,4-dichloro-indigo whiteFigure 36. Chemical reaction of NBT and BCIP substrates with AP. BCIP is hydrolyzed by alkaline phosphatase to form an intermediate that undergoes dimerization to produce an indigo dye. The NBT is reduced to the NBT-formazan by the two reducing equivalents generated by the dimerization. This reaction proceeds at a steady rate, allowing accurate control of the relative sensitivity and control of the development of the reaction.Pierce Metal Enhanced DAB Substrate KitThermo Scientific Pierce Metal Enhanced DAB SubstrateFeatures:Kit uses cobalt and nickel chloride in a special formulation Superior sensitivity50 times more sensitive than the of diaminobenzidine peroxidase substrate that yieldstraditional DAB method and 30 times more sensitive than intense color for immunohistochemical staining using HRP. other metal-intensified versions of DAB Low background, high intensityget a crisp, dark brown-black precipitate that is more intense than the dull brown precipitate when using DAB without enhancement; even with the increased intensity, background is almost nonexistent Only two componentssimply mix the two liquid components and your working solution is ready to use 6-hour stabilitythe innovative working solution is stable for more than 6 hours at room temperature, while other DAB substrates must be used immediately56'