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para-Nitrophenylphosphate (pNPP) is a non-proteinaceous chromogenic substrate for alkaline and acid phosphatases used in ELISA and conventional spectrophotometric assays. [1] Phosphatases catalyze the hydrolysis of pNPP liberating inorganic phosphate and the conjugate base of para -nitrophenol (pNP).
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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 ...
4-Nitrophenol is a product of the enzymatic cleavage of several synthetic substrates such as 4-nitrophenyl phosphate (used as a substrate for alkaline phosphatase), 4-nitrophenyl acetate (for carbonic anhydrase), 4-nitrophenyl-β-D-glucopyranoside and other sugar derivatives which are used to assay various glycosidase enzymes.
Other names in common use include nitrophenyl phosphatase, p-nitrophenylphosphatase, para-nitrophenyl phosphatase, K-pNPPase, NPPase, PNPPase, Ecto-p-nitrophenyl phosphatase, and p-nitrophenylphosphate phosphohydrolase. This enzyme participates in γ-hexachlorocyclohexane degradation.
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).
Phosphoric monoester hydrolases (or phosphomonoesterases) are enzymes that catalyse the hydrolysis of O-P bonds by nucleophilic attack of phosphorus by cysteine residues or coordinated metal ions.
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 .