Thermo Scientific Pierce DAB and the DAB Substrate Kit enable chromogenic detection of horseradish peroxidase (HRP) activity based the action of 3,3'-diaminobenzidine (DAB) in Western blot and tissue staining methods.
DAB (3,3'-diaminobenzidine tetrahydrochloride; MW = 214.1) reacts with HRP in the presence of peroxide to yield an insoluble brown-colored product at locations where peroxidase-conjugated antibodies are bound to samples. The brown precipitate is insoluble in alcohol and other organic solvents, making it an excellent substrate for immunohistochemical staining that requires the use of traditional counterstains and mounting media. The Pierce DAB Substrate Kit includes a 10X solution of DAB and Stable Peroxide Substrate Buffer. Crystalline DAB (powder) is also available.
- HRP substrate – for detection of horseradish peroxidase activity on solid media, including nitrocellulose and PVDF membranes and fixed tissue samples
- Chromogenic – no special equipment needed for visualization; the DAB reaction produces brown bands or spots at sites of reaction with HRP-conjugated antibodies or probes
- Package options – choose powder for custom formulation with buffer and peroxide or a complete kit of preformulated solutions for immediate use
Highlights of Pierce DAB Substrate Kit (Part No. 34002):
- Easy-to-use – dilute 10X stock solution to 1X in provided stable peroxide buffer
- Safer – preformulated DAB solution minimizes safety concerns with this chemical
- Complete – includes convenient solution of stable hydrogen peroxide buffer (Part No. 34062)
- Stable – store refrigerated at 4°C for at least one year; freezer storage is not necessary
DAB has been used in a variety of applications, including Western blotting, immunohistochemistry and electron microscopy. Many enhancement methods have been reported for DAB involving different buffer conditions, the addition of metal ions and post-treatment applications (Ref. 1-7; also see the Pierce Metal Enhanced DAB Substrate Kit, Part No. 34065). One disadvantage of DAB compared to other substrates is the need to use the working solution immediately because it begins a reaction process upon the addition of hydrogen peroxide. This results in increased background color of the substrate. However, the brown bands resulting from DAB reactions do not fade as quickly as the color-products of other precipitating HRP substrates.
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Immunohistochemistry (IHC) technical guide
IHC immunodetection technical guide
Pierce Stable Peroxide Buffer (10X)
Metal Enhanced DAB Substrate Kit