2-D Electrophoresis 二維電泳操作教學 - 銘傳大學.pptVIP

2-D Electrophoresis二維電泳操作教學 前言 第1章　二維電泳(2-D Electrophoresis)簡介 Core technique of proteomics Capable of simultaneously resolving thousands of proteins in one separation procedure Immobilized pH gradient gels: higher resolution improved interlaboratory reproducibility higher protein loading capacity extended basic pH limit Sorts proteins according to two independent properties in two discrete steps: the first-dimension step, isoelectric focusing(IEF), separates proteins according to their isoelectric pints(pI); the second-dimension step, SDS-polyacrylamide gel electrophoresis(SDS), separate proteins according to their molecular weights. Each spot on the resulting two-dimensional array corresponds to a single protein species in the sample Thousands of different proteins can thus be separated, and information such as the protein pI, the apparent molecular weight, and the amount of each protein is obtained Experimental sequence for 2-D electrophoresis Sample preparation IPG strip rehydration IEF IPG strip equilibration SDSVisualization Analysis 圖一　Ettan? IPGphor? Isoelectric Focusing System Ettan IPGphor Isoelectric Focusing System Ceramic manifold Electrode assembly Rehydration tray Pre-cut electrode wicks Spirit level IPG Cover Fluid Cleaning solution Cleaning brush 第3章　樣品的製備 The 2-D process begins with sample preparation Appropriate sample preparation is absolutely essential for a good 2-D result Keep the sample preparation strategy as simple as possible to avoid protein losses Preserve sample quality by preparing the sample just prior to IEF or storing samples in aliquots at -80°C 第4章 IPG Strip Rehydration (IPG 膠條覆水作用） 先將rehydration tray (覆水槽) 的蓋子打開，首先以ddH2O徹底沖洗覆水槽的凹槽，滴入數滴的Ettan IPGphor Strip Holder Cleaning Solution (清潔液)，用Cleaning brush (清潔刷子)仔細刷洗凹槽，移除任何可能的殘留蛋白質，再用ddH2O沖洗乾淨後，徹底風乾。 依照使用者手冊上的指示，配製適當體積的rehydration solution (覆水試劑) ，依照兩組蛋白質樣本濃度的比例分別和lysis buffer混合而成；接著加入適當體積的IPG Buffer。 利用Spirit level (水平儀) 來測量rehydration tray的放置是水平的。 將配好的rehydration