蛋白质纯化解答.ppt

Two Dimensional Electrophoresis of E. coli Proteins - more than 2,000 proteins were visualized 三、蛋白纯化原则Guidelines for Protein Purification 确定目标 purity, activity and quantity 充分了解目的蛋白及主要杂质的理化特性 to simplify technique selection and optimisation 确定活性测定方法 fast detection of protein activity/recovery and critical contaminants 尽量减少纯化步骤 extra steps reduce yield and increase time, combine steps logically 纯化目的确定纯化目标 三、蛋白纯化原则Guidelines for Protein Purification 确定目标 purity, activity and quantity 充分了解目的蛋白及主要杂质的理化特性 to simplify technique selection and optimisation 确定活性测定方法 fast detection of protein activity/recovery and critical contaminants 尽量减少纯化步骤 extra steps reduce yield and increase time, combine steps logically ASSAY: A method that measures the amount of a specific protein, in a mixture of others. If possible, the assay should give an estimate of the absolute concentration (e.g. mg/ml). Relative concentration is the next best. The method chosen will ultimately depend on the properties of the target protein. Most common are: enzymatic assays. ligand-binding assays immunological assays 活性测定方法的特点：快速、简便、特异和定量 确定活性检测方法 三、蛋白纯化原则Guidelines for Protein Purification 确定目标 purity, activity and quantity 充分了解目的蛋白及主要杂质的理化特性 to simplify technique selection and optimisation 确定活性测定方法 fast detection of protein activity/recovery and critical contaminants 尽量减少纯化步骤 extra steps reduce yield and increase time, combine steps logically 尽量减少纯化步骤 Guidelines for Protein Purification 减少每步操作时间 to avoid lengthy procedures which risk losing activity/reducing recovery 减少添加物 additives may need to be removed in an extra purification step or may interfere with activity assays 早期除去降解物 for example, proteases 每步使用不同的纯化方法 to take advantage of sample characteristics which can be used for separation (size, charge, hydrophobicity, ligand specificity) KEEP IT SIMPLE! 四、蛋白质的鉴定 测定蛋白质的浓度 凯氏定氮法 紫外吸收法 分光光度法 测定蛋白质的纯度 鉴定目标蛋白质 蛋白质分子质量与等电点的测定 氨基酸组成及顺序分析 蛋白质结晶与结构分析 蛋白质活性及功能测定 1 蛋白质含量的测定 凯氏定氮法： 蛋白质平均含氮量为16﹪，首先测定氮的含量，进