(完整版)westernblot原理及操作
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Part3: Troubleshooting
A.High background
B.No/Weak signal
Part1: Experiment Principle
➢ The immobilization of proteins onto a membrane, and the subsequent detection by proteinspecific binding and detection reagents
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 samples 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
Joule Heating
Q=W=PT=UIT=
非纯电阻电路:Q<W
resistance Heat
inconsistent field strength and transfer lose its buffering capacity
cause the gel to melt/ stick to the membrane/条带出现波浪状
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 staining
C. Transfer of proteins and staining
Two basic electrical equations ➢ Ohm’s law： V = I x R (The resistance is inherent in the system) ➢ Power equation: P = I x V = I2 x R = V2/R
Electrophoresis
➢ 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）柱状（管状）电泳： 3．pH的连续性： （1）连续pH电泳 （2）非连续pH电泳
➢ 阳性对照 ➢ Marker
✓ enable the determination of the protein size ✓ monitor the progress of an electrophoretic run
➢ 内参 未染色(pre mixed) 预染Marker(pre-stained): 单色预染、多色预染
Electrophoresis
➢ 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
Β-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 steps
A. Sample preparation B. Electrophoresis C. . Transfer of proteins and staining D.Detection
A. Sample preparation
Denaturing detergent/离子型 ：SDS, deoxycholate Less 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.
➢ Western blotting identifies with specific antibodies proteins that have been separated from one another according to their size by gel electrophoresis.
Western Blot
catalogue
Part1: Experiment Principle Part2: Operating steps
A. Sample preparation B. Electrophoresis C. Transfer of proteins and staining D.Detection
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 thoroughly
缓冲液的离子强度 Check the pH; it should be around 8.3
Transfer buffer 有效地从胶上洗脱蛋白，将蛋白结合到膜上
Tris: 保持导电性、缓冲体系的PH
促进SDS从蛋白上洗脱
methanol 促进蛋白结合到膜上
SDS
降低蛋白从胶中洗脱
High qualityotherwise High conductivity
ຫໍສະໝຸດ Baidu
Poor transfer
Glycerol ：increase the density of the sample to be loaded Bromophenol blue: a small anionic dye molecule During protein sample treatment the sample should be mixed by vortexing before and after the heating step for best resolution
A.High background
B.No/Weak signal
Part1: Experiment Principle
➢ The immobilization of proteins onto a membrane, and the subsequent detection by proteinspecific binding and detection reagents
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 samples 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
Joule Heating
Q=W=PT=UIT=
非纯电阻电路:Q<W
resistance Heat
inconsistent field strength and transfer lose its buffering capacity
cause the gel to melt/ stick to the membrane/条带出现波浪状
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 staining
C. Transfer of proteins and staining
Two basic electrical equations ➢ Ohm’s law： V = I x R (The resistance is inherent in the system) ➢ Power equation: P = I x V = I2 x R = V2/R
Electrophoresis
➢ 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）柱状（管状）电泳： 3．pH的连续性： （1）连续pH电泳 （2）非连续pH电泳
➢ 阳性对照 ➢ Marker
✓ enable the determination of the protein size ✓ monitor the progress of an electrophoretic run
➢ 内参 未染色(pre mixed) 预染Marker(pre-stained): 单色预染、多色预染
Electrophoresis
➢ 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
Β-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 steps
A. Sample preparation B. Electrophoresis C. . Transfer of proteins and staining D.Detection
A. Sample preparation
Denaturing detergent/离子型 ：SDS, deoxycholate Less 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.
➢ Western blotting identifies with specific antibodies proteins that have been separated from one another according to their size by gel electrophoresis.
Western Blot
catalogue
Part1: Experiment Principle Part2: Operating steps
A. Sample preparation B. Electrophoresis C. Transfer of proteins and staining D.Detection
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 thoroughly
缓冲液的离子强度 Check the pH; it should be around 8.3
Transfer buffer 有效地从胶上洗脱蛋白，将蛋白结合到膜上
Tris: 保持导电性、缓冲体系的PH
促进SDS从蛋白上洗脱
methanol 促进蛋白结合到膜上
SDS
降低蛋白从胶中洗脱
High qualityotherwise High conductivity
ຫໍສະໝຸດ Baidu
Poor transfer
Glycerol ：increase the density of the sample to be loaded Bromophenol blue: a small anionic dye molecule During protein sample treatment the sample should be mixed by vortexing before and after the heating step for best resolution