导图社区 ST-Dayne
这是一篇关于ST-Dayne的思维导图
编辑于2022-02-21 13:19:13ST-Dayne
植物总蛋白提取
200g甜菊叶在液氮研磨,500mL缓冲液((50 mM KPi pH 8.0, 5.0 mM MgCl2, 1.0 mM EDTA, 10 mM DTT, 10 g PVPP, 10 g PVP, and protease inhibitor cocktail)。
聚乙烯吡咯烷酮。聚乙烯吡咯烷酮是一种非离子型高分子化合物,PVP是以单体乙烯基吡咯烷酮(NVP)
过滤去除不溶性物质,18000g,离心30min。
硫酸铵(30-75%饱和度)沉淀可溶性物质,15000g,15min收集。
溶解(buffer B:50 mM HEPES pH 8.0, 5.0 mM MgCl2, 1.0 mM EDTA and 10 mM DTT)
DEAE column (1.5 × 20 cm, Aogma Biosciences)纯化:BC30 (buffer B with 30 mM NaCl) to BC250 (buffer B with 250 mM NaCl)
10-kDa浓缩管浓缩
与探针冰上孵育
竞争组:全蛋白1mg/ml,100 ul 与10 uM的ST-Dayne孵育+底物:steviol
The proteome (1 mg/ml, 100 L) were incubated with ST-Dayne (10 M) in the presence or absence of free steviol on ice.
对照组:全蛋白1mg/ml,100 ul 与10 uM的ST-Dayne孵育-底物
The proteome (1 mg/ml, 100 L) were incubated with ST-Dayne (10 M) in the presence or absence of free steviol on ice.
点击化学反应
96孔板,用365nm紫外光,冰上照射20min
20 uL 反应样品+0.25 uL/tube(含1% SDS): TAMRA-N3 (10 mM stock in DMSO,Lumiprobe), CuSO4 (100 mM stock in H2O), THPTA (Tris(3-hydroxypropyltriazolylmethyl)amin, (10 mM stock in H2O) NaVc (100 mM stock in H2O).
室温孵育1 h
SDS-PAGE
上样20 uL