520 组织蛋白酶 D 检测试剂盒 荧光法、520 Cathepsin D Assay Kit *Fluorimetric*详细如下: 520 组织蛋白酶 D 活性检测试剂盒使用 QXL™ 520/HiLyte™ Fluor 488 标记的 FRET 肽底物来测量酶活性。在完整的 FRET 肽中,HiLyte™ Fluor 488 的荧光被 QXL™ 520 淬灭。在 FRET 肽被活性酶切割后,可以在激发/发射 = 490 nm/520 nm 下连续监测荧光的增加。HiLyte™ Fluor 488 的荧光信号在低 pH 值下稳定,这对于组织蛋白酶活性是最佳的。该试剂盒可用于检测生物样品和纯化酶制剂中组织蛋白酶D酶的活性。检测限:0.78 ng/ml 组织蛋白酶是一类球状溶酶体蛋白酶,在哺乳动物细胞更新中起着至关重要的作用。它们降解多肽并通过它们的底物特异性来区分。组织蛋白酶 D 是溶酶体天冬氨酸蛋白酶,在细胞内蛋白质分解中具有活性。 Note: Warm all kit components until thawed to room temperature before starting the experiments. Prepare assay buffer: Prepare fresh assay buffer for each experiment. Refer to Table 1. Cathepsin D substrate solution: Dilute Cathepsin D substrate (Component A) 100-fold in assay buffer. Prepare fresh assay buffer for each experiment. Refer to Table 2. Cathepsin D diluent: Dilute the enzyme (Component C) 1:400 in assay buffer. This amount of enzyme is enough for a full 96-well plate. If not using the entire plate, adjust the amount of enzyme to be diluted accordingly. Pepstatin A (Cathepsin D inhibitor): Dilute the 100 uM inhibitor solution (Component E) 1:1000 in assay buffer. The diluted pepstatin solution has a concentration of 100 nM. Add 10 ul of the diluted pepstatin into each of the inhibitor control well. Add test compounds and diluted enzyme solution to the microplate wells. The suggested volume of enzyme solution for a 96-well plate is 40 uL/well and test compound is 10 uL/well. Simultaneously set up the following control wells, as deemed necessary: Positive control contains the enzyme without test compound. Inhibitor control contains Cathepsin D enzyme and pepstatin. Vehicle control contains Cathepsin D enzyme and vehicle used in delivering test compound (e.g. DMSO, concentration not to exceed 1%). Test compound control contains assay buffer and test compound. Some test compounds have strong autofluorescence and may give false results. Substrate control contains assay buffer. Using the assay buffer, bring the total volume of all controls to 50 uL. Optional: Pre-incubate the plate for 10 min. at assay temperature. Any temperature (the assay temperature) from room temperature to 37 ℃ may be used, as long as the subsequent incubations are performed at the same temperature.