westernblot原理及操作课件

1、Western BlotPart1: Experiment PrinciplePart2: Operating stepscatalogueA. Sample preparation B. Electrophoresis C. Transfer of proteins and staining. D.Detection Part3: Troubleshooting A.High background B.No/Weak signal Part1: Experiment PrincipleWestern blotting identifies with specific antibodies p

2、roteins that have been separated from one another according to their size by gel electrophoresis. The immobilization of proteins onto a membrane, and the subsequent detection by protein-specific binding and detection reagents 显微电泳自由界面电泳区带电泳1.支持物的物理性状：（1）纸电泳（2）粉末电泳（3）凝胶电泳（4）缘线电泳2支持物的装置形式：（1）平板式电泳（2）垂

3、直板电泳（3）柱状（管状）电泳：3pH的连续性：（1）连续pH电泳（2）非连续pH电泳ElectrophoresisElectrophoresis电荷分子量SDS阴离子去污剂蛋白质：SDS-1:1.4破坏蛋白质的二级和三级结构-巯基乙醇/DTT强还原剂破坏蛋白质的四级结构带负电荷的蛋白质-SDS复合物蛋白质-SDS胶束Part2: Operating stepsA. Sample preparation B. Electrophoresis C. Transfer of proteins and staining. D.Detection A. Sample preparation Denat

4、uring detergent/离子型 ：SDS, deoxycholateLess denaturing detergent/非离子型 : Triton x-100, NP-40短期4保存，长期-20保存2. Protease and phosphatase inhibitors As soon as lysis occurs, proteolysis, dephosphorylation and denaturation begin. 4. Determination of protein concentration 变性解聚/氧化还原暴露抗原表位（表位可能位于3D构象的内部）5. Pre

5、paration of samples for loading into gels Loading bufferVolumeAmount: 30-50ug 过犹不及，内参粘连，荧光灼烧式淬灭Glycerol ：increase the density of the sample to be loadedBromophenol blue: a small anionic dye molecule During protein sample treatment the sample should be mixed by vortexing before and after the heating

6、step for best resolution B.Electrophoresis 交联剂催化剂助凝剂使每个蛋白的起跑线相同，提高解析度Acry Pore size 高浓度胶小分子量低浓度胶大分子量液封 ：沿玻板流下，避免产生气泡；动作轻慢 0.5-1h凝胶 Clean thoroughlyCheck the pH; it should be around 8.3 缓冲液的离子强度阳性对照Markerenable the determination of the protein size monitor the progress of an electrophoretic run内参未染色(

7、pre mixed)预染Marker(pre-stained): 单色预染、多色预染纯化好的蛋白混合物，与染料共价偶联选择宽范围，条带分布均匀的Marker内参：确保等量上样C. Transfer of proteins and stainingC. Transfer of proteins and stainingTwo basic electrical equations Ohms law： V = I x R (The resistance is inherent in the system) Power equation: P = I x V = I2 x R = V2/R Joule

8、 Heating Q=W=PT=UIT=非纯电阻电路:QNP-40Tween20）8.一抗、二抗的稀释比例过低9.二抗不是非特异性结合：与封闭剂反应， Antibody-Specific Ligands 9.孵育装置被污染10.孵育温度过高11.曝光时间过长A.High background B.No/Weak signal 1.无效上样：抗原量不足、蛋白质降解、目标蛋白表达低2.抗原在电泳和转膜的过程中变性：热敏感蛋白注意系统散热3.转膜不充分：预先在甲醇中浸泡 电极正确装配、降温、优化转膜电流及时间4.胶和膜位置放反5.膜的错误选择6.过度封闭：降低浓度、减少封闭时间7.没有足够的一抗、二抗与目标蛋白结合：降低稀释比例，延长孵育时间（过犹不及）8.一抗失活：保存条件，避免反复冻融 洗涤剂能够影响抗体的结合 使用蒸馏水9.二抗失活：一抗中有NaN3，一抗后洗膜要充分 用硫柳汞钠作二抗的抑菌剂10.一抗、二抗不匹配11.曝光时间过短12.底物量不足13.检测系统缺乏足够的灵敏度：确保蛋白的上样量在检测范围内Mutiple bands1.蛋白样品有