第7章 蛋白质及分离、纯化和表征.ppt

第7章　蛋白质的分离、纯化和表征 Protein Separation, Purification and Characterization 对蛋白质分子进行的各种研究，要建立在对蛋白质分子本身的性质（特别是区别于其它生物与化学分子的独特的性质）和各种实验手段的原理和适用性深入了解的基础上。 Protein Purification and Characterization The aim of protein purification is to isolate one particular protein from all the others in the starting material, exploits利用: ?Solubility ?Size ? Charge ?Hydrophobicity or/and specific binding affinity of the protein of interest Ionization of protein 蛋白质的滴定曲线形状和等电点，在有中性盐存在下可以发生明显的变化。 蛋白质等电点在一定程度上决定于介质中离子的组成。 没有其他盐类干扰时，蛋白质质子供体基团解离出来的质子数与质子受体基团结合的质子数相等时的pH称为等离子点(isoionic point)。 沉降分析法测定相对分子质量 离心机 转速 60 000-80 000rpm 离心力 400 00- 700 000g 沉降分析法测定相对分子质量 离心力 4π2 v 2 r F = g F :离心力，单位以地心引力的倍数g表示（即g 或×g） g :重力加速度，等于980.6 cm2/s2. r : 通常指自离心管中轴底部内壁到离心转轴中心之间的距离（cm） v :为转速，即离心机每秒的转数，若以每分钟转数（rpm）表示，则上式应为 4π2 v 2 r F = （1/60）2 g Gel filtration chromatography Size exclusion chromatography Molecular sieve（筛子） chromatography ?Molecules are separated on the basis of their size and shape. The protein sample in a small volumes is applied to the top of a column of porous(有孔的) beads that are made of an insoluble but highly hydrated （亲水的）polymer such as polyacrylamide (Bio-Gel) or the carbohydrates dextran （葡聚糖 ）(Sephadex) or agarose (琼脂糖) (Sepharose) ? Small molecules can enter the pores in the beads whereas larger or more elongated molecules cannot. The smaller molecules therefore have a larger volume of liquid accessible （可接近的） to them; both the liquid surrounding the porous heads and that inside the beads. ? In contrast, the larger molecules have only the liquid surrounding the beads accessible to them, and thus move through the column faster, emerging out of the bottom (eluting洗脱) first. ? The smaller molecules move more slowly through the column and elute later. Electrophoresis of Protein In polyacrylamide(聚丙烯酰胺) gel electrophoresis (PAGE) proteins are applied to a porous