Western Blot.ppt

Western Blot 配制: 5ml10% 分离胶 3ml5% 积层胶 ddH 2O 30%Acr 20%SDS 10%AP TEMED 灌制分离胶隔绝空气 ddH 2O :<8% 异丁醇: >8% 灌制积层胶插入梳子 Western blot (immunoblotting) 1. What is Western blot? A technique for detecting specific proteins separated by electrophoresis by use of labeled antibodies . So called since it has some similarity to a Southern blot. 2. Why we have to use it? We can use this technique to identify a target protein in plex mixture, and we can also use it to measure it ’ s expression level . 3. How to do it? 步骤: ? Separate proteins by gel electrophoresis ? Transfer proteins onto a membrane – Wet tank transfer system – Semi-dry transfer system ? Identify proteins – Immunodetection 优点: ?高分辨率的电泳技术?特异敏感的抗原-抗体反应?1-5 ng 中等大小的靶蛋白蛋白提取? Total protein – standard lysis buffer ( lipa ) ? Nuclear cytoplasmic extraction (NER-PER, extraction kit, Pierce) ? To prevent degradation always centrifuge samples at 4 ℃ and include a protease inhibitor ? Protein Qantification : Standard BCA assay (Pierce) Bradford reagent SDS-PAGE 不连续电泳? Poly Acrylamide Gel Electrophoresis ( PAGE ) is the best method in protein separate 。? Use PAGE to separate proteins by MW 。 SDS : ?阴离子去污剂变性剂?氨基酸侧链与 SDS 充分结合形成带负电荷的蛋白质-SDS 胶束。?蛋白质-SDS 胶束所带的负电荷大大超过了蛋白质分子原有的电荷量,消除了不同分子之间原有的电荷差异。?与强还原剂一起使蛋白分子氢键、疏水键打开,使蛋白质分子线性化。