生物化学 Exploring Proteins(讲稿).pptx

1§5.1样品纯化的基本流程§5.2层析（Chromatography）§5.3电泳（Electrophoresis）§5.4肽的测序与合成SequencingSynthesis§5.5免疫印迹与ELISA§5.6质谱（MS）与蛋白质研究§5.7蛋白质3D结构的测定§5ExploringProteins

2§5.1样品纯化的基本流程TissueHomogenization(匀浆)Filtration(过滤）Crudeextract（粗提品）desalination(脱盐)Concentration(浓缩)ColumnchromatographySaltingOut(盐析）Centrifugation(离心)(Cultured)CellCelllysis可使用电泳分析每步的分离效果和最终样品的纯度

3GeneralCentrifugation

4Ultracentrifugation:separationandmassdeterminationm:themassoftheparticle;v:thepartialspecificvolumeρ:thedensityofthemedium;f:thefrictionalcoefficient(ameasureoftheshapeoftheparticle).

5SaltingOutAmmoniumsulfateisthemostpopularsaltused

6Dialysis

7Concentration高分子量聚乙二醇（PEG）

8§5.1样品纯化的基本流程§5.2层析（Chromatography）§5.3电泳（Electrophoresis）§5.4肽的测序与合成SequencingSynthesis§5.5免疫印迹与ELISA§5.6质谱（MS）与蛋白质研究§5.7蛋白质3D结构的测定§5ExploringProteins

9Chromatography:separatingsubstancesbyallowingthemtopartitionbetweentwophases,onemobile,onestationary.Differencesincharge,size,Affinity,hydrophobicinteractions,chirality(smallmolecule)canbeexploitedforsubstanceseparationwithchromatography.§5.2层析（Chromatography）

10Ion-exchangechromatographySize-exclusionchromatographyAffinitychromatographyHydrophobicinteractionchromatographyChromatography

11Ion-exchangechromatography

12线性盐浓度梯度的形成

13Size-exclusion(Gel-filtration)chromatographymassdetermination

14Affinitychromatography

15HydrophobicInteractionChromatography

17§5.1样品纯化的基本流程§5.2层析（Chromatography）§5.3电泳（Electrophoresis）§5.4肽的测序与合成SequencingSynthesis§5.5免疫印迹与ELISA§5.6质谱（MS）与蛋白质研究§5.7蛋白质3D结构的测定§5ExploringProteins

18Principleofelectrophoresis电泳原理与粒子大小、形状有关的系数粒子所带电荷大小带电粒子的泳动能力带电粒子的移动速度电场强度

19MediaofelectrophoresisMedia:polyacrylamidegel（聚丙烯酰胺凝胶）Socalled:polyacrylamidegelelectrophoresis,PAGE

20丙烯酰胺亚甲基（甲叉）双丙烯酰胺Formingofpolyacrylamidegel

21Nativee