样品实验方案
溶液配制
储备溶液配制
1.Covidyte TF670储备液(200X):向Covidyte TF670小瓶中添加25 µL(对于cat#13540)或250 µL(对于cat#13541)DMSO。
注意:制成单次使用的等分试样,并储存在-20°C下。
工作溶液配制
1.Covidyte TF670工作溶液:在20 mM Tris缓冲液(pH 7.5)或自备缓冲液中以1:200稀释底物储备液。每个实验在96孔板中使用50μL底物溶液。
2.冠状病毒蛋白酶稀释:根据需要稀释冠状病毒蛋白酶。
实验步骤
(一个96孔板的样品方案)
- 将50μLCovidyte TF670工作溶液和50μL冠状病毒蛋白酶稀释液添加到测定板的所有孔中。
- 使用荧光酶标仪在Ex / Em = 640/680 nm(截止660nm)处检测荧光的增加。
对于动力学读数:立即开始连续不断地测量荧光强度,并每5分钟记录一次数据,持续30-120分钟。
对于终点读数:在避光的条件下,将反应在所需温度下孵育30至120分钟。然后测量荧光强度。
图示
图1蛋白酶在蛋白质激活、细胞调节和信号转导以及氨基酸的生成中起着至关重要的作用,用于蛋白质合成或其他代谢途径。 FRET蛋白酶底物广泛用于检测蛋白酶活性,特别是用于病毒蛋白酶的检测,这些病毒蛋白酶经常需要较长的肽序列才能实现最佳结合,例如冠状病毒,HIV和HCV蛋白酶。原理:内部淬灭的FRET肽底物被蛋白酶消化,荧光强度与蛋白酶活性成正比,从而产生高荧光的肽片段。Tide Quencher、Tide Fluor 和iFluor 染料都是研发用于高通量筛选应用的FRET蛋白酶底物的极其有效的淬灭剂。
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相关产品
品牌 |
品名 |
货号 |
规格 |
描述 |
AAT |
Covidyte IF670 |
#13542 |
100 Tests |
1.包含可被冠状病毒蛋白酶切割的14个氨基酸序列的肽底物
2.#13542:当该肽被冠状病毒蛋白酶水解时,iFluor 670片段产生明显增强的荧光,因为其荧光不再被TQ5淬灭;#13541:当该肽被冠状病毒蛋白酶水解时,TF5片段产生明显增强的荧光,因为其荧光不再被TQ5淬灭
|
AAT |
Covidyte TF670 |
#13541 |
1000 Tests |
AAT |
Covidyte EN450 |
#13535 |
100 Tests |
AAT |
Covidyte ED450 |
#13537 |
100 Tests |
包含可被冠状病毒蛋白酶切割的11个氨基酸序列的肽底物 |
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