武汉云克隆科技股份有限公司

Tryptase is a serine protease with trypsin-like activity, which is sometimes also referred to as Mast Cell Protease 7. It is stored in the secretory granules of mouse mast cells. It exhibits anticoagulant activity due to its ability to degrade fibrinogen in the presence of the diverse array of protease inhibitors in plasma. The activity of recombinant human TPS is measured by its ability to cleave a fluorogenic peptide substrate Mca-Arg-Pro-Lys-Pro-Val-Glu-Nval-Trp-Arg-Lys(Dnp)-NH2 in the assay buffer 50 mM Tris, pH 8.5. The rhTPS is diluted to 200 ug/ml in 50 mM Tris, 150 mM NaCl, 10 mM CaCl2, 0.05% (w/v) Brij-35, pH 7.5, then activated with 0.1 ug/ml Thermolysin at 37 ℃ for 15min followed by adding 10 mM 1, 10 phenanthroline to stop activation. The activated rhTPS is diluted to 50 ug/mL in heparin incubation buffer of 100 µg/mL heparin, 50 mM MES, pH 5.5 and incubated at room temperature for 2 hours. Then the rhTPS was diluted to 12.5 ug/ml in assay buffer and load into a black well plate 50 µL and start the reaction by adding 50 µL of 20 µM substrate, with a substrate blank containing 50 µL assay buffer, 50 µL substrate, and no rhTPS. Then read at excitiation and emission wavelengths of 320 nm and 405 nm, respectively, in kinetic mode for 5 minutes. The specific activity of recombinant human TPS is > 25 pmol/min/µg.