westernblot原理及操作课件.ppt

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 Principle Western blotting identifies with specific antibodies proteins

2、 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）垂直板电泳（3）

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 Denaturing

4、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.Preparation of sampl

5、es for loading into gels Loading buffer Volume Amount: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 u During protein sample treatment the sample should be mixed by vortexing before and after the heating step for best re

6、solution 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(pre mixed)预染Mar

7、ker(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 Heating Q=W=PT=UIT=非纯电阻电路:QN

8、P-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.蛋白样品有多种修饰形式2.蛋白降解3.一抗、二抗浓度过高：非特异性结合4.抗体未纯化5.蛋白多聚体形成（迁移太快，温度过高）Detection of phosphorylated protein1.蛋白酶、磷酸酶抑制剂2.保证一抗孵育时间3.选用好的磷酸化抗体4.BSA封闭和稀释