AMC 组织型 (tPA) 活性检测试剂盒 荧光法

库存 ：大量

英文名 ：AMC tPA Activity Assay Kit *Fluorimetric*

CAS号 ：AMC tPA Activity Assay Kit *Fluorimetric*

保质期 ：详见说明

供应商 ：齐源生物

保存条件 ：-20°C

规格 ：1 kit

AMC 组织型 (tPA) 活性检测试剂盒 荧光法、AMC tPA Activity Assay Kit *Fluorimetric*详细如下：
AMC tPA 活性检测试剂盒针对检测 tPA 酶活性进行了优化。该试剂盒包含具有高反应性和低背景的荧光底物。tPA 裂解底物，导致 AMC（7-氨基-4-甲基香豆素）荧光团的释放。可以在激发/发射= 354/442 nm 处监测荧光。该试剂盒可用于高通量筛选 tPA 诱导剂和抑制剂。
组织型纤溶酶原激活剂 (tPA) 是一种丝氨酸蛋白酶，可将酶原纤溶酶原切割成活性纤溶酶。已经认识到tPA在各种生理和病理过程中起重要作用。
1X Assay buffer: Add 5 ml of 2X assay buffer (Component C) to 5 mL deionized water.
tPA substrate solution: Dilute tPA substrate (Component A) 1:100 in 1X assay buffer  according to Table 1. For each experiment, prepare fresh substrate solution.
tPA diluent: Dilute tPA enzyme (Component D) 1:100 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.
tPA inhibitor (Leupeptin): Add 10 ul of the 10 mM inhibitor solution (Component E) into each of the inhibitor control well of a 96-well plate.
Add test compounds and diluted enzyme solution to the microplate wells. For one well of a 96-well plate, the suggested volume of enzyme solution is 40 uL and 10 uL of test compound.
Simultaneously establish the following control wells, as deemed necessary:
Positive control contains the tPA without test compound.
Inhibitor control contains tPA and control inhibitor.
Vehicle control contains tPA and vehicle used in delivering test compound (e.g. DMSO, concentration not to exceed 1%).
Test compound control contains 1X assay buffer and test compound. Some test compounds have strong autofluorescence and may give false results.
Substrate control contains 1X assay buffer.
Using the 1X 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.
Add 50 uL of tPA substrate solution from Step 1.2 into each well. For best accuracy, it is advisable to have the substrate solution equilibrated to the assay temperature. Mix the reagents completely by shaking the plate gently for 30 sec.
For kinetic reading: Immediately start measuring fluorescence intensity at Ex/Em=354 /442 nm continuously and record data every 5 min for 30 to 60 min.
For end-point reading: Incubate the reaction for 30 to 60 min. Keep plate from direct light. Mix the reagents and measure fluorescence intensity at Ex/Em=354/442 nm.