细胞裂解液中加入抗目的蛋白的抗体.PPTVIP

免疫共沉淀技术原理 Co-Immunoprecipitation(CO-IP) a classical method used for research protein-protein interaction on the basis of specificity of antibody to antigen. an effective method to confirm two kinds of proteins endogenous interaction in cell. It utilizes antibody to precipitate the corresponding specific molecule meanwhile the other molecules with specific binding to the molecule will be precipitated. be used to validate proteins specific binding. 免疫共沉淀技术原理 基本原理是：在非变性条件下，将细胞裂解，细胞裂解液中加入抗目的蛋白的抗体，与抗原形成特异免疫复合物，经过洗脱，收集免疫复合物，然后进行SDS及Western blotting分析. 免疫共沉淀技术原理图解 hCLP46与calnexin相互作用研究方法 CO-IP预实验 CO-IP操作步骤 Western Blotting检测calnexin与hCLP46的相互作用 Western Blotting检测calnexin与hCLP46的相互作用 用12%SDS胶对蛋白样品进行电泳，每孔上样20μL。分离后，200mA电转至PVDF膜,上。含5%脱脂奶粉的TBST(20mmol/L Tris pH7.6,150mmol/LNaCl,0.1% tween-20)室温封, 闭PVDF膜1h。含3%脱脂奶粉的TBST按1:2000, 稀释Myc抗体，按1:100稀释calnexin抗体。 4℃杂交过夜。TBST洗膜3次，每次10min,含 5%脱脂奶粉的TBST按1:2000稀释偶联HRP的二抗，室温杂交1h TBST洗膜3次，每次10min，TBST再洗膜3次，每次5min,Millipore 发光液按1:1混合，滴在膜上显色10s,暗室曝光。 结果分析 裂解液中各成分浓度对免疫共沉淀的影响 1: hCLP46 input; 2: calnexin input; 3: IP anti-Myc 沉淀 未检测到沉淀中存在calnexin． Fig.1 Damage of high concentrations of salt and partial alkaline environment to interaction between hCLP46 and calnexin 结果分析 抗体用量对免疫共沉淀的影响 1: hCLP46 input; 2: calnexin input; 3: IP control，293Trex 细胞做对照; 4: IP anti-Myc 沉淀，未检测到沉淀中存在calnexin． Fig.2 No interaction between hCLP46 and calnexin with low concentration of antibodies 结果分析 抗体用量对免疫共沉淀的影响 1: hCLP46 input; 2: calnexin input; 3: IP control，293Trex 细胞做对照; 4: IP anti-Myc 沉淀，检测到沉淀中存在calnexin． Fig.3 Interaction between hCLP46 and calnexin after exalting the dosage of antibody 结果分析 hCLP蛋白量对免疫共沉淀的影响 1: hCLP46 input; 2: calnexin input; 3: IP anti-Myc 沉淀，检测到沉淀中存在hCLP46; 4: IP anti-Myc 沉淀，未检测到沉淀中存在calnexin Fig.4 Damage of too high hCLP46 expression to interaction between hCLP46 and calnexin 结果分析 交联剂DSP对免疫共沉淀的影响 1: hCLP46 input; 2: calnexin input; 3: IP anti-Myc 沉淀，检测到沉淀中存在hCLP46; 4: IP control，293Trex