疏水作用色谱和共价色谱——高等生物分离技术.ppt

* The seminar is built up around four major questions, that I will try to answer: What is HIC? What can it be used for? Interaction and separation principles How to do it? * 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 the protein is incorrectly folded or damaged. This picture shows hydrophobic and hydrophilic parts on the surface of lysozyme. The most hydrophobic parts are dark red, the less hydrophobic lighter red. The most hydrophilic parts are shown in dark blue, with the less hydrophilic parts are lighter blue. -疏水相互作用层析是根据它们的疏水性分离的生物分子的液相的色谱技术。 虽然大多数疏水性氨基酸被埋藏在球状蛋白的内部，但其中的一些被暴露在蛋白质的表面上的疏水性氨基酸HIC-介质相互作用。不同蛋白质暴露的疏水氨基酸的疏水能力不同的。 此图为溶菌酶的表面上的疏水和亲水部分。最疏水部分为暗红色，少疏水浅红色。最亲水部分示于深蓝色，具有较少的亲水性部分是淡蓝色。 * * Why should I use HIC? Complementary to other techniques, IEX, GF and affinity Mild: HIC is a mild, non-denaturing method. Natural follow up to ammonium sulphate precipitation, since it is promoted by moderately high concentrations of neutral salts, which also has a stabilizing effect on on protein structure. High selectivity, can differentiatebetween small differences in hydrophobicity High recovery: normally achieve recoveries above 75% Relatively high capacity, Phenyl Sepharose 6 FF high sub: 30 mg IgG/ml gel and 36 mg HSA/ml gel Concentrating: large sample volumes of dilute samples can be applied, followed by elution in a much smaller volume 我为什么要使用HIC？ 补充其他技术，IEX，GF和亲和层析 温和：HIC是一种温和的，非破坏性非变性的方法。当适当提高中性盐的浓度，其自然随着硫酸铵沉淀，其中也有对蛋白质结构的稳定性有一定的