Alkaline phosphatase tissue-nonspecific (ALPL) is a membrane-bound glycoprotein expressed in bone, liver, kidney and other tissues. It corely mediates skeletal and dental mineralization, regulates vitamin B6 metabolism and adaptive thermogenesis. Mutations in its gene cause hypophosphatasia, leading to defective skeletal mineralization.This method is based on the principle that alkaline phosphatase can catalyze the hydrolysis of p-nitrophenyl phosphate (PNPP) to produce p-nitrophenol, which has a maximum absorption peak at 405 nm, and the activity of alkaline phosphatase is determined by quantifying the generated p-nitrophenol. First, perform 2-fold serial dilution of the protein starting from 100 μg/mL to prepare 7 gradient concentrations with an additional buffer control group, then dilute the PNPP substrate to 20 mM with the assay buffer (50 mM Tris, 1 mM MgCl2, pH 9.0), add 50 μL of the diluted protein sample and 50 μL of 20 mM PNPP substrate to each well of the microplate, and conduct kinetic absorbance reading at 405 nm for 5 consecutive minutes. For the preparation of the standard curve, first dilute the p-nitrophenol standard to 1 mM with the assay buffer, then make 2-fold serial dilutions to obtain 7 gradient concentrations, add 100 μL of each standard dilution to the corresponding wells, set a blank control well with only 100 μL of assay buffer, and finally measure the absorbance of all wells at 400 nm.<br/>One unit of enzyme activity is defined as the 1 μg of enzyme required to hydrolyzes 1pmol PNPP to produce PNP in 1 min at 37°C. The specific activity of recombinant cattle ALPL is 1146 pmol/min/μg.<br/>Specific Activity (pmol/min/μg)=<br/>△OD=Adjusted for Substrate Blank<br/>F=Conversion Factor(convert from standard curve of p-nitrocatechol)<br/>T= Time<br/>N=Amount of enzyme