520 颗粒酶 A 活性检测试剂盒,荧光法、520 Granzyme A Activity Assay Kit, Fluorimetric详细如下: 520 Granzyme A Assay Kit 为筛选酶抑制剂或使用新型内部猝灭的 5-FAM/QXL® FRET 底物检测酶活性提供了方便的检测方法。在被颗粒酶 A 切割后,FRET 底物将被分成两个片段,导致释放 5-FAM 荧光,该荧光可以在激发/发射 = 490 nm/520 nm 下进行监测。5-FAM 的长波长荧光受生物样品和测试化合物中成分的自发荧光的干扰较小。本试剂盒可用于检测生物样品和纯化酶制剂中颗粒酶A的活性。 粒酶是结构相关的丝氨酸蛋白酶家族。它们储存在细胞毒性淋巴细胞的细胞毒性颗粒中。已知颗粒酶可诱导靶细胞凋亡。 粒酶 A 是细胞毒性 T 淋巴细胞 (CTL) 和自然杀伤 (NK) 细胞分泌的最丰富的丝氨酸蛋白酶。Granzyme A 通过穿孔素途径进入含有病原体的细胞和肿瘤细胞,并诱导不依赖于半胱天冬酶的细胞凋亡。因此,对半胱天冬酶或粒酶 B(一种主要的凋亡介质)具有抗性的细胞可能对粒酶 A 敏感。最近的研究表明,粒酶 A 激活了一种从线粒体开始的新的程序性细胞死亡途径。Granzyme A 还针对几种重要的核蛋白进行降解,包括组蛋白和 DNA 损伤修复蛋白。粒酶 A 具有诱导某些细胞因子分泌的促炎活性。 Add test compounds and granzyme A enzyme solution (step 1.3) to the microplate wells. The suggested volume of enzyme solution for one well of a 96-well plate is 40 µL and test compound is 10 µL. Establish the following control wells at the same time, as deemed necessary: Positive control: Add 40 µL granzyme A enzyme solution (step 1.3) and 10µL 1X assay buffer (step 1.1). Inhibitor control: Add 40 µL granzyme A enzyme solution (step 1.3) and 10µL granzyme A inhibitor (step 1.4). Vehicle control: Add 40 µL granzyme A enzyme solution (step 1.3) and 10µL vehicle used in delivering test compound (e.g. DMSO, concentration not to exceed 1%). Test compound control: Add 40µL 1X assay buffer (step 1.1) and 10µL test compound. Some test compounds have strong autofluorescence and may give false results. Substrate control: Add 50µL 1X assay buffer (step 1.1). The total volume of all controls should be 50 µL. Optional: Pre-incubate the plate for 10 min. at assay temperature. Any temperature (the assay temperature) from room temperature to 37°C may be used, as long as the subsequent incubations are performed at the same temperature.