lishufeng-高级生化实验.ppt

2. In terms of solubility isoelectric precipitation Salting in and salting out Organic solvent precipitation Salting in / Salting out Salting IN low concentrations of salt usually increases the solubility of charged macromolecules. Salting OUT high concentrations of added salt lowers the solubility of and they come out of solution. Effect of K2SO4 on the solubility of Hb-CO 离子强度 溶解度 1) electrophoresis电泳 charged proteins move toward the opposite electrode. Many kinds of different electrophoresis methods 3 . Based on charge difference 聚丙烯酰胺凝胶电泳 等电聚焦/isoelectric focusing, IEF 2-dimensional electronphoresis 2、 Ion-exchange chromatography 离子交换层析法 Separates proteins on the basis of charge Two kinds: Anion/阴离子-exchange Cation/阳离子-exchange Ion-exchange chromatography Methods for elution. changing pH and/or increasing ionic strength. Changes done by either stepwise or gradient 4.Affinity Chromatography 亲和层析 specific binding of protein enzyme substrate IgG protein A antigen antibody Ni-matrix (His)6 GST GSH Elution Mechanism shift to acidic pH (usually to pH 2-4) in the form of a step gradient. Elution with specific ligands or analog 层析的分类 1）离子交换层析 2）凝胶过滤层析 3）亲和层析 4）吸附层析 5）分配层析 6）疏水层析 1. Protein concentration assay Total protein： Kjedahl nitrogen assay/凯氏定氮法 Lowry method/Folin-酚法 BCA Assay Biuret assay/双缩脲法 UV absorption/紫外吸收法 Specific protein content: Activity assay, western blot 蛋白浓度、纯度及活性测定 2. purity Electrophoresis: single band HPLC: single peak Solubility analysis crystallization 3.Activity analysis 4. Storage of protein keeping purified protein in 50% glycerol,? or? 1% serum albumin? at -70? -80o C? or liquid nitrogen if needed Store in small portions , avoid repeated freeze-thaw. Protein Purification Disrupt Blender, homoginizer Remove debris Centrifugation Precipitate/concentrate Ammonium sulfate Purify Chromatography Quality Analysis: Activity, purity, concentration Storage * * * * 质粒 M 双 双 单 单 15,000 10,000 7,500 5,00