3,3'-Diaminobenzidine
Encyclopedia
3,3'-Diaminobenzidine is an organic compound
. This derivative of benzidine
is a precursor to polybenzimidazole fiber
. As its water-soluble tetrahydrochloride
, it has been used in immunohistochemical staining of nucleic acid
s and proteins.
and ammonia
with a copper catalyst at high temperature and pressure, followed by acidic workup.
in the presence of hemoglobin
to give a dark-brown color. This is used to detect fingerprints in blood. Improperly prepared tissue samples may give false positives
In research, this reaction is used to stain cells that were prepared with hydrogen peroxidase
enzyme, following common immunocytochemistry
protocols. In research being done on Alzheimer's Disease
, Aβ protein amyloid plaques are targeted by a primary antibody, and subsequently by a secondary antibody, which is conjugated with a peroxidase
enzyme. This will bind DAB as a substrate and oxidize it, producing an easily observable brown color. Plaques can then be quantified for further evaluation. One other method uses complexes of injected biocytin
with avidin
or streptavidin
, biotin, and then peroxidase.
Organic compound
An organic compound is any member of a large class of gaseous, liquid, or solid chemical compounds whose molecules contain carbon. For historical reasons discussed below, a few types of carbon-containing compounds such as carbides, carbonates, simple oxides of carbon, and cyanides, as well as the...
. This derivative of benzidine
Benzidine
Benzidine, the trivial name for 4,4'-diaminobiphenyl, is the solid organic compound with the formula 2. This aromatic amine is a component of a test for cyanide and also in the production of dyes...
is a precursor to polybenzimidazole fiber
Polybenzimidazole fiber
Polybenzimidazole fiber is a synthetic fiber with a very high melting point that also does not readily ignite, because of its exceptional thermal and chemical stability. The U.S...
. As its water-soluble tetrahydrochloride
Hydrochloride
In chemistry, hydrochlorides are salts resulting, or regarded as resulting, from the reaction of hydrochloric acid with an organic base . This is also known as muriate, derived from hydrochloric acid's other name: muriatic acid....
, it has been used in immunohistochemical staining of nucleic acid
Nucleic acid
Nucleic acids are biological molecules essential for life, and include DNA and RNA . Together with proteins, nucleic acids make up the most important macromolecules; each is found in abundance in all living things, where they function in encoding, transmitting and expressing genetic information...
s and proteins.
Preparation
Diaminobenzidine, which is commercially available, is prepared by reacting 3,3'-dichlorobenzidine3,3'-Dichlorobenzidine
3,3'-Dichlorobenzidine is used in the production of azo dyes and is considered a carcinogen. It contains two benzene rings. This compound has been shown to increase the incidence of tumors in animals. Because it is structurally similar to benzidine, a known carcinogen, it is believed that it may...
and ammonia
Ammonia
Ammonia is a compound of nitrogen and hydrogen with the formula . It is a colourless gas with a characteristic pungent odour. Ammonia contributes significantly to the nutritional needs of terrestrial organisms by serving as a precursor to food and fertilizers. Ammonia, either directly or...
with a copper catalyst at high temperature and pressure, followed by acidic workup.
Applications
Diaminobenzidine is oxidized by hydrogen peroxideHydrogen peroxide
Hydrogen peroxide is the simplest peroxide and an oxidizer. Hydrogen peroxide is a clear liquid, slightly more viscous than water. In dilute solution, it appears colorless. With its oxidizing properties, hydrogen peroxide is often used as a bleach or cleaning agent...
in the presence of hemoglobin
Hemoglobin
Hemoglobin is the iron-containing oxygen-transport metalloprotein in the red blood cells of all vertebrates, with the exception of the fish family Channichthyidae, as well as the tissues of some invertebrates...
to give a dark-brown color. This is used to detect fingerprints in blood. Improperly prepared tissue samples may give false positives
In research, this reaction is used to stain cells that were prepared with hydrogen peroxidase
Peroxidase
Peroxidases are a large family of enzymes that typically catalyze a reaction of the form:For many of these enzymes the optimal substrate is hydrogen peroxide, but others are more active with organic hydroperoxides such as lipid peroxides...
enzyme, following common immunocytochemistry
Immunocytochemistry
Immunocytochemistry is a common laboratory technique that uses antibodies that target specific peptides or protein antigens in the cell via specific epitopes. These bound antibodies can then be detected using several different methods. ICC allows researchers to evaluate whether or not cells in a...
protocols. In research being done on Alzheimer's Disease
Alzheimer's disease
Alzheimer's disease also known in medical literature as Alzheimer disease is the most common form of dementia. There is no cure for the disease, which worsens as it progresses, and eventually leads to death...
, Aβ protein amyloid plaques are targeted by a primary antibody, and subsequently by a secondary antibody, which is conjugated with a peroxidase
Peroxidase
Peroxidases are a large family of enzymes that typically catalyze a reaction of the form:For many of these enzymes the optimal substrate is hydrogen peroxide, but others are more active with organic hydroperoxides such as lipid peroxides...
enzyme. This will bind DAB as a substrate and oxidize it, producing an easily observable brown color. Plaques can then be quantified for further evaluation. One other method uses complexes of injected biocytin
Biocytin
Biocytin is a chemical compound that is an amide formed from the vitamin biotin and the amino acid L-lysine. As an intermediate in the metabolism of biotin, biocytin occurs naturally in blood serum and urine....
with avidin
Avidin
Avidin is a tetrameric biotin-binding protein produced in the oviducts of birds, reptiles and amphibians deposited in the whites of their eggs. In chicken egg white, avidin makes up approximately 0.05% of total protein...
or streptavidin
Streptavidin
Streptavidin is a 60000 dalton protein purified from the bacterium Streptomyces avidinii. Streptavidin homo-tetramers have an extraordinarily high affinity for biotin . With a dissociation constant on the order of ≈10-14 mol/L, the binding of biotin to streptavidin is one of the strongest...
, biotin, and then peroxidase.