ST-Dayne

这是一篇关于ST-Dayne的思维导图

ST-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