金属螯合层析MCAC_XP1答案.pptVIP

第七章 金属螯合层析 一、基本原理 金属螯合层析 Metal Chelate Chromatography， MCC 又称为固定化金属离子吸附层析 Immobilized Metal Ion Adsorption Chromatography, IMAC 金属螯合层析是利用不同蛋白质分子表面所含组氨酸的差别而将其分离纯化的一种层析技术。 金属螯合层析的介质通常是在惰性支持物上接有固定化的金属离子，组氨酸与金属离子具有螯合作用，对于具有不同组氨酸含量的不同蛋白质分子对于一定金属离子的亲和力大小不同 ，因而可通过柱层析进行分离。 二、金属螯合剂 在Sepharos上接有亚氨基二乙酸 Iminodiacetic acid , IDE 将氯化锌或硫酸铜等溶液通过该柱时即可制备出锌或铜等金属螯合剂。 IDE Na+ 三、影响吸附的因素 1. 与金属离子的性质有关 Cu2+、Zn2+、Ca2+、Ni2+、 Mg2+等 2. 与样品的性质有关 His含量不同 His分布位置不同 Buffer 的 pH 及 离子强度 四、实验方法 20mM, pH8.0 PBS+0.5M NaCl + CuSO4 平衡Buffer平衡 上样 改变 pH 或 离子强度洗脱 50mM EDTA 再生除去金属离子 Affinity-tagged fusion proteins The affinity-tag binds to a specific ligand Only the tagged fusion protein binds to the ligand Binding usually possible in 8 M Urea or 6 M GuHCl (depends on tag) A protease cleavage site allows the tag to be removed after purification Purity typically 90% in one step r-Protein Cleavage site Specific ligand Matrix Affinity tag Target protein Affinity tag Ligand Glutathione-S-Transferase (GST) Glutathione Oligo(Histidine) Nickel ions E tag sequence Anti-E antibody ZZ (domain B of protein A) IgG Protein A IgG Recombinant fusion proteins Deliberately designed for affinity purification Note: Buffers may include 8 M urea or 6 M GuHCl when purifying proteins solubilised from inclusion bodies. Elution with Competing free ligand GST-fusion proteins on Glutathione Sepharose Column: Glutathione Sepharose 4B, pre-packed Binding: PBS + 1% Triton X-100 Elution: 5 mM Glutathione, 50 mM Tris.HCl, pH 8.0 Purification and Detection of (His)6 Fusion Proteins r-Prot. His His His Ni2+ His His His His Ni2+ Ni2+ Ni2+ Elution with Competing free binding substance Oligo(His)-fusion proteins on Chelating Sepharose Column: HiTrap Chelating, Ni2+ Binding: 20 mM phosphate, 0.5 M NaCl, 10 mM Imidazole Elution: As binding buffer except 500 mM Imidazole Note: Buffers may also include 8 M urea or 6 M GuHCl when purifying proteins solubilised from inclusion bo