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The enzyme alkaline phosphatase (ALP, alkaline phenyl phosphatase, also abbreviated PhoA) is a phosphatase with the physiological role of dephosphorylating compounds. The enzyme is found across a multitude of organisms, prokaryotes and eukaryotes alike, with the same general function, but in different structural forms suitable to the environment they function in. Alkaline phosphatase is found ...
To allow detection of the target protein, the secondary antibody is commonly linked to biotin or a reporter enzyme such as alkaline phosphatase or horseradish peroxidase. This means that several secondary antibodies will bind to one primary antibody and enhance the signal, allowing the detection of proteins of a much lower concentration than ...
5-Bromo-4-chloro-3-indolyl phosphate (BCIP, X-phosphate, XP) is an artificial chromogenic substrate used for the sensitive colorimetric detection of alkaline phosphatase activity. It is, for example, used in immunoblotting , in situ hybridization , and immunohistochemistry , often in combination with nitro blue tetrazolium chloride (NBT).
In immunohistochemistry the alkaline phosphatase is often used as a marker, conjugated to an antibody. The colored product can either be of the NBT/BCIP reaction reveals where the antibody is bound, or can be used in immunofluorescence .
The Bodansky unit is an obsolete measure of alkaline phosphatase concentration in blood. It is defined as the quantity of alkaline phosphatase that liberates 1 mg of phosphate ion during the first hour of incubation with a buffered substrate containing sodium β-glycerophosphate. [1]
Enzymes used in ELISAs include horseradish peroxidase (HRP), alkaline phosphatase (AP) or glucose oxidase. These enzymes allow for detection often because they produce an observable color change in the presence of certain reagents. In some cases these enzymes are exposed to reagents which cause them to produce light or chemiluminescence.
For example, for the protein alkaline phosphatase, the staining solution is a mixture of 4-chloro-2-2methylbenzenediazonium salt with 3-phospho-2-naphthoic acid-2'-4'-dimethyl aniline in Tris buffer. This stain is commercially sold as a kit for staining gels.