上游充通大学 1.The Purification of Proteins SHANGHAI JIAO TONG UNIVERSITY lon-exchange chromatography Low salt Medium High salt Protein mixture buffer salt buffer buffer Negatively Elution charged bead column Highly Negatively positively charged charged protein protein Less Collect positively fractions charged protein Negatively Positively charged charged
1. The Purification of Proteins lon-exchange chromatography
上游充通大兽 1.The Purification of Proteins SHANGHAI JIAO TONG UNIVERSITY 3)利用蛋白质亲和性差异进行分离 Affinity chromatography(亲和层析) 生物大分子的某些特定结构能够同其他分子相互识别并结 合，这种结合是特异的又是可逆的，生物分子间的这种结 合能力称为亲和力。 抗原抗体 激素-受体 Protein to be purified Mixture of (e.g.,insulin receptor) Ligand protein (e.g.,insulin) sought ( and contaminant() Agarose bead
1. The Purification of Proteins 3）利用蛋白质亲和性差异进行分离 Affinity chromatography（亲和层析） 生物大分子的某些特定结构能够同其他分子相互识别并结 合，这种结合是特异的又是可逆的，生物分子间的这种结 合能力称为亲和力。 抗原-抗体 激素-受体
上游充通大学 1.The Purification of Proteins SHANGHAI JIAO TONG UNIVERSITY Affinity chromatography(亲和层析) 操作简便，特异性强；价格昂贵 1-Incubate crude sample with the immobilized ligand 3-Elute 2-Wash away non bound sample components from solid support
1. The Purification of Proteins Affinity chromatography（亲和层析） 操作简便，特异性强；价格昂贵
上游充通大兽 1.The Purification of Proteins SHANGHAI JIAO TONG UNIVERSITY Affinity chromatography(亲和层析) ©HiS6:6个连续组氨酸 重组蛋白常用亲和标签 ©GST:谷胱甘肽转移酶 CBD:几丁质结合结构域(Chitin Binding Domain) MBP:麦芽糖结合蛋白(Maltose Binding Protein) Biotinated peptide:生物素化多肽链 NH His protein
His6：6个连续组氨酸 GST：谷胱甘肽转移酶 CBD：几丁质结合结构域(Chitin Binding Domain) MBP：麦芽糖结合蛋白(Maltose Binding Protein) Biotinated peptide：生物素化多肽链 1. The Purification of Proteins Affinity chromatography（亲和层析） 重组蛋白常用亲和标签 NH2 NH2 Ni His His His His protein
上游充通大兽 1.The Purification of Proteins SHANGHAI JIAO TONG UNIVERSITY 常压纯化装置： Sample injection port 蠕动泵 Solvent Pump reservoir 层析柱 检测器 Column 记录仪 分部收集器 Detector Chart recorder 紫外检测仪 Fraction collector ATo waste
常压纯化装置： 蠕动泵 层析柱 检测器 记录仪 分部收集器 紫外检测仪 1. The Purification of Proteins