北京大学.生物化学.2005.ppt

Selenocysteine - the 21st amino acid Prokaryotic selenocysteine incorporation    Working with proteins Methods for separating proteins take advantage of properties such as charge, size, and solubility, which vary from one protein to the next. Because many proteins bind to other biomolecules, proteins can also be separated on the basis of their binding properties. Column chromatography  Ion-exchange chromatography Size-exclusion chromatography Affinity chromatography . . . . . .  谢谢大家! 蛋白质可以通过各种生物化学技术纯化 利用蛋白质的溶解度、净电荷、大小以及与配体结合特异性上的微小差异。有透析、凝胶过滤、离子交换层析、亲和层析、电泳（垂直板电泳、等电聚焦电泳、双向电泳）等分离纯化方法。透析 Salting out and dialysis(透析) Ion-exchange Chromatography 离子交换 分离氨基酸常用的是带有耐酸性非常强的磺酸根SO3－Na＋（以盐的形式出现）的强阳离子交换树脂。首先将这种树脂填充到柱子中，然后注入含有样品的流动相，样品中含有阳离子成分X＋，通过静电吸引，与树脂中的带电基团相互作用，结果X＋与Na＋交换，即发生阳离子交换后，形成SO3－X＋。 Size-exclusion Chromatography 分子筛 This method separates proteins according to size. The column contains a cross-linked polymer with pores of selected size. Larger proteins migrate faster than smaller ones, because they are too large to enter the pores in the beads and hence take a more direct route through the column. The smaller proteins enter the pores and are slowed by the more labyrinthian path they take through the column. Affinity Chromatography 亲和层析 Affinity chromatography separates proteins by their binding specificities. The proteins retained on the column are those that bind specifically to a ligand cross-linked to the beads. (In biochemistry, the term ligand is used to refer to a group or molecule that is bound. ) After nonspecific proteins are washed through the column, the bound protein of particular interest is eluted by a solution containing free ligand. Figure 1-10 Figure 1-11 Figure 1-10 Figure 1-10 Figure 1-18 Figure 1-19 Figure 1-10 Figure 1-18 Figure 1-18 3.1.3 Nonstandard amino acids 4－羟(基)脯氨酸 5－羟(基)赖氨酸 硒代半胱氨酸 链霉素 6－N－甲基赖氨酸 γ －羟基谷氨酸 鸟氨酸 瓜氨酸 About 300 kinds of a. a. not occurring in proteins Se