520 FRET Sirt1 检测试剂盒 荧光法、520 FRET SIRT1 Assay Kit *Fluorimetric*详细如下: 520 FRET SIRT1 Assay Kit 提供了一种方便的两步均相程序,用于使用 FRET 底物测量 sirtuin 1 活性。在第一步中,乙酰化底物与含有 SIRT1 的样品一起孵育。底物的去乙酰化首先使其敏感,因此在第二步中与 SIRT 显影剂混合会产生荧光团,然后可以在 Ex/Em=490/520 nm 处检测到。该试剂盒可用于 SIRT1 抑制剂/激活剂的高通量筛选。FRET底物的长波长荧光受细胞成分和测试化合物的自发荧光的干扰较小。核提取物 HDAC(包含 HDAC 1 和 2)不会使 FRET sirtuin 底物去乙酰化。测定以标准的 96 孔格式进行。检测限:13 ng/ml 组蛋白去乙酰化酶 (HDAC) 酶通过组蛋白上赖氨酸残基的去乙酰化作为基因的转录抑制因子。Sirtuins (SIRT) 是一类独特的脱乙酰酶 (III 类 HDAC),需要 NAD 作为辅因子。Sirtuin 1 (SIRT1) 参与调节肿瘤抑制因子 p53 和抑制细胞凋亡。 SIRT1 FRET substrate solution: Dilute SIRT1 substrate (Component A) and NAD+ (Component E) in assay buffer (Component D). Both SIRT1 substrate and NAD+ should be diluted in assay buffer 100-fold. For each experiment, prepare fresh substrate solution. SIRT1, human recombinant enzyme (Component C): ready to use. For positive control, use 40 µL of Component C per well. 1X developer: Dilute the developer (Component G) and the nicotinamide (Component F) in assay buffer (Component D). Both developer and nicotinamide should be diluted 10-fold in assay buffer. Each well requires 50 uL of developer solution. Add test compounds and SIRT1 enzyme to the microplate wells. For one assay in a 96-well plate, the suggested volume of enzyme solution is 40 uL and 10 uL of test compound. Establish the following control wells at the same time, as deemed necessary: Positive control contains SIRT1 enzyme without test compound. Inhibitor/activator control contains SIRT1 enzyme and SIRT1 inhibitor/activator. Vehicle control contains SIRT1 enzyme and vehicle used in delivering test compound (e.g. DMSO, concentration not to exceed 1%). Test compound control contains assay buffer (Component D) and test compound. Some test compounds may themselves be fluorescent and thereby give false results. Substrate control contains assay buffer (Component D). Using the assay buffer (Component D), bring the total volume of all controls to 50 uL. Pre-incubate the plate for 10 min at 37 ℃.