NBT Assay Protocols

NBT or nitro blue tetrazolium is a chemical compound used in biochemistry and immunology for detecting the enzyme alkaline phosphatase. The substance is also used in the diagnosis test of chronic granulomatous disease, which affects the immunodefense cells. NBT assay protocols include immunoblotting, BCIP/NBT alkaline phosphatase substrate solution and detection of galactosidase and alkaline phosphatase activities in tissue.

Immunoblotting

• Immunoblotting is a technique used to detect antibodies, which are proteins involved in the immunodefense process, says Susan R. Haynes in "RNA-Protein Interaction Protocols." This includes the use of NBT, as well as bovine serum albumin, primary antibodies 9E10, Comassie staining solution, nitrocellulose membranes, Tris base buffer solution, ammonium per-sulfate, sodium dodecyl sulfate and water-saturated buthanol.

BCIP/NBT Alkaline Phosphatase Substrate Solution

• Technicians prepare a solution with three drops of 1 percent 5-Bromo-4-chloro-3-indolyl phosphate (BCIP), three drops of 1.5 percent NBT, two drops of the chemical substance Levamisole and alkaline phosphatase (AP) buffer at pH 9.5, according to the IHC World website. The solution is then incubated at room temperature and available to use.

Detection of Galactosidase and Alkaline Phosphatase Activities in Tissue

• Galactosidases are enzymes that can break the galactoside molecule, present in the bacteria Escherichia coli and other organisms. According to Protocol Online, during this protocol, biologists use rat cell samples treated with PBS solution, glutaraldehyde and paraformaldyde. Then, they prepare the bromo-chloro-indolyl-galactopyranoside or X-gal reaction buffer for the samples, using potassium ferrocyanide, magnesium chloride, sodium deoxycholate and the detergent Nonidet P-40. Technicians can use nitro blue tetrazolium (NBT) instead of potassium ferrocyanide as a faster alternative. Cell samples are later observed under microscope.