520 去泛素化检测试剂盒 荧光法、520 Deubiquitination Assay Kit *Fluorimetric*详细如下: 520 去泛素化检测试剂盒为筛选酶抑制剂和激活剂或通过使用荧光肽底物连续检测去泛素化活性提供了一种方便的检测方法。在被去泛素蛋白酶切割后,该底物产生绿色荧光团,可以在激发/发射 = 490/520 nm 下检测到。 去泛素化酶 (DUB) 是一种能逆转泛素修饰的蛋白酶,可以使蛋白质免于蛋白酶体降解。DUB 特异性切割泛素最后一个残基 (Gly76) 末端羰基下游的泛素连接分子。他们分为五个家族。定义明确的家族包括泛素羧基末端水解酶 (UCH) 和泛素加工蛋白酶 (UBP)。 Note: Bring all kit components until thawed to room temperature before starting the experiments. Prepare 1X assay buffer: Prepare fresh DTT-containing 1X assay buffer for each experiment. Use this DTT-containing assay buffer in all the following steps. If not using the entire plate, adjust the amount of assay buffer to be diluted accordingly. The 520 DUB substrate solution: Dilute deubiquitin substrate (Component A) 1:100 in 1X assay buffer. Refer to Table 2. For each experiment, prepare fresh substrate solution. If not using the entire plate, adjust the amount of substrate to be diluted accordingly. Protease diluents: Dilute the enzyme, UCH-L3 (Component C), 1:200 in 1X 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. Note: Prepare enzyme diluents immediately before use. Do not vortex the enzyme solution. Prolonged storage or vigorous agitation of the diluted enzyme will cause denaturation. Store the enzyme solution on ice. UCH-L3 inhibitor: Dilute the 4 mM inhibitor solution (Component F) 1:10 in 1X assay buffer to get a concentration of 0.4 mM. Add 10 ul of the diluted inhibitor into each of the inhibitor control well.