Application of 3,3'-Diaminobenzidine (DAB)

3,3'-Diaminobenzidine (DAB) is an organic compound with the formula (C6H3(NH2)2)2. This derivative of benzidine is a precursor to polybenzimidazole, which forms fibers that are renowned for their chemical and thermal stability [1]. 3,3'-Diaminobenzidine is oxidized in the presence of peroxidase and hydrogen peroxide resulting in the deposition of a brown, alcohol-insoluble precipitate at the site of enzymatic activity. DAB produces a dark brown reaction product. DAB is used in the staining of nucleic acids and proteins, most commonly for immunohistochemical procedures. Although it is water soluble in its unoxidized form, it forms a water-insoluble brown precipitate when oxidized. In immunohistochemical procedures, the location of proteins can be detected using DAB as a substrate. A protein of interest is targeted by an antibody that is conjugated with a peroxidase enzyme, and in the presence of hydrogen peroxide, DAB is readily catalyzed to its oxidized form, forming a brown precipitate [2]. DAB is commercially available. It is prepared by treating 3,3'-dichlorobenzidine with ammonia with a copper catalyst at high temperature and pressure, followed by acidic workup [3]. An advantage of DAB chromogenic staining is that the colored precipitate formed during the reaction between HRP and DAB is not sensitive to light and the slides can be stored for many years.

DAB is symmetric about the central carbon bond between both ring structures. In the crystal, the rings of each molecule are co-planar and the amine units connect molecules to form an intermolecular 3-dimensional hydrogen bond network [4, 5].

3,3'-Diaminobenzidine is a mutagenic compound and should be handled with care, using gloves and appropriate protective gear. Unused compound can be disposed of as a hazardous waste or neutralized through a simple chemical procedure. Presumed to have carcinogenic potential for humans. DAB is classified as a suspected carcinogen with the potential of producing irreversible effects. However, there is evidence suggesting that although 3,3'-diaminobenzidine is structurally related to benzidine, it may not exhibit a similar carcinogenic effect.

3,3'-Diaminobenzidine Immunohistochemistry Substrate is supplied as a two components liquid substrate system designed for the rapid and simple detection of peroxidase in immunohistochemical and in situ hybridization procedures. The two components consist of 3,3'-Diaminobenzidine Immunohistochemistry Concentrate (Reagent A) and DAB Immunohistochemistry Reaction Buffer (Reagent B) which is a ready to use reaction buffer containing hydrogen peroxide.

DAB is a widely used chromogen for immunohistochemical staining and immunoblotting. When in the presence of peroxidase enzyme, DAB produces a brown precipitate that is insoluble in alcohol and xylene. Cardassian DAB comes in a 3-component system consisting of a liquid stable 3,3'-Diaminobenzidine Chromogen, 3,3'-Diaminobenzidine (DAB) Substrate Buffer and DAB Enhancer. The enhancer adds contrast and a slight increase in staining intensity.

References

[1] H. Schwenecke, D. Mayer, "Benzidine and Benzidine Derivatives" in Ullmann’s Encyclopedia of Industrial Chemistry, 2005, Wiley-VCH, Weinheim.
[2] H. Roschzttardtz, L. Grillet, M.-P. Isaure, G. Conejero, R. Ortega, C. Curie, S. Mari, The Plant Cell Nucleolus as a Hot Spot for Iron, The Journal of Biological Chemistry, 2011, 286 (32): 27863–27866.
[3] US 3943175, Druin, Melvin L. & Oringer, Kenneth, "Synthesis of pure 3,3'-diaminobenzidine", issued 1976-03-09, assigned to Celanese Corporation.
[4] H. F. Qian, W. Huang, Biphenyl-3,3',4,4'-tetraamine. Acta Crystallographica., 2010, E66 (5): o1060. doi:10.1107/S1600536810012511.
[5] https://en.wikipedia.org/wiki/3,3%27-Diaminobenzidine.

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