3-2层析基础课稿.pptVIP

分子筛的 优点 有 操作 简单，一个缓冲液走到底 并且对缓冲液没有特殊要求 用分子筛的同时可以置换缓冲液 他是一种非吸附的技术，条件温和样品回收率高 不同填料有特定的分离范围 柱效怎么测？ 离子交换层析（IEX） 离子交换层析是一种吸附层析,根据带电荷的分子和层析填料上相反电荷之间可逆的相互作用进行分离. 离子交换层析是基于分子之间电荷的差异进行分离. 离子交换填料上也是带有电荷的 阴离子交换填料 阴离子带负电荷 阴离子交换剂是带正电荷的配基，结合带负电荷的分子，替换样品溶液中的阴离子 最常见的配基有 Q, DEAE, ANX 阳离子交换填料 阳离子带正电荷 阳离子交换剂是带负电荷的配基，结合带正电荷的分子，替换样品溶液中的阳离子 最常见的配基有 S, SP, CM 弱离子交换剂: 载量随 pH 而变化 什么是疏水层析? 疏水层析是一种液相吸附层系，它是依据生物分子之间的疏水性的差别来进行分离的。 组成蛋白质的氨基酸有的是疏水性质的，所以蛋白质上具有一些疏水区域。 改变缓冲液条件就可以使蛋白解离 疏水层析通常使用的条件 含有高盐的结合缓冲液: 50 mM PBS pH 7.0 ,含有 1-1.5 M 硫酸铵 低盐的洗脱缓冲液: 50 mM PBS pH 7.0 梯度: 10-15 柱体积 流速: 参考说明书 填料: Phenyl Sepharose? 6 Fast Flow (high sub) 亲和层析是一种通过生物分子之间的特异性的相互作用来分离物质的层析方法.如酶和底物、抗原与抗体的非常专一的相互作用。将配基固定在填料上，就可以结合相应的生物分子，再采用合适的方法使之解离 为什么选择亲和层析? 亲和层析能够完成一般方法很难完成的分离 如变性和非变性的，功能不同的蛋白 经常可以一步达到 90 % 的纯度 快速将目标蛋白与大量的杂质分离 速度快，操作简单 Strong ion exchangers retain their charge A strong ion exchange group remains charged over a very wide range of pHs. This is a good thing: an ion exchanger can only work if it has charged groups so we dont want to lose them. Preparation of the sample Preparation of the sample for the chromatographic step means to transfer it to a suitable buffer and adjust the concentration of protein. Volume The volume of the sample is less critical. However, the time taken to apply the sample can be critical, since certain proteins are labile and may loose the activity under a prolonged exposure to the ion exchanger. Hydrophobic interaction chromatography is a liquid chromatography technique that separates biomolecules according to their hydrophobicity. Although most hydrophobic amino acids are buried in the interior of globular proteins, some of them are exposed on the proteins surface. The ligands on HIC-gels interact with the exposed hydrophobic amino acids on the surface of the proteins. The amount of exposed hydrophobic amino acids differ between proteins. HIC is even sensitive enough to be influenced by non-polar groups normally buried within the interior of the protein, but exposed if