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> 分子筛
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> 2021年6月24日 19:45
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- 凝胶过滤层析（又称分子筛、分子排阻）
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- 构成
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◊ 柱形：瘦高
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◊ 填料：多孔球状基架
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◊ 颗粒直径范围：8.6\~200μm
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◊ 材质：交联的多糖
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◊ 图 片
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◊
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- 分离原理
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◊ 根据分子大小和形状的差异进行分离的一种简单可靠的层析技术
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◊ 图 片
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◊
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- 分离特点
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◊ 分子量、形状、体积大小影响进孔能力及出峰体积
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◊ 大分子先出，小分子后出
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◊ 提高选择性和柱效
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◊ 只需要一种缓冲液
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◊ 容易操作
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◊ 凝胶过滤
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◊ 脱盐置换缓冲液
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◊ 体积排阻
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◊ 分子量相差2倍以上，分离效果才会好
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◊ 蛋白质溶液脱盐
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◊ 添加到150mM NaCl的盐到流动相中以消除分子和填料间的作用对结果的干扰
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◊ 蛋白复性
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- 层析步骤
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◊ 平衡→上样→洗脱→清洗/保存
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- 层析图谱
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- 相关特性
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◊
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样品的分子量Mr与出峰体积Ve一一对应
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> ◊
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> ◊
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> 样品分子量Mr决定样品的入孔能力系数Kd
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> ◊
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> ◊ 样品分子量Mr与出峰体积Ve（入孔能力Kav）之间的关系
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> ◊
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> 凝胶过滤层析填料的分离范围：a值
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> ◊
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> ◊
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> 凝胶过滤层析填料的分辨率（选择性）：b值
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> ◊
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> ◊ 凝胶过滤层析填料的a值和b值的拟合测定
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> ◊
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- 凝胶过滤层析的条件优化（影响因素）
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◊ 填料的孔径与选择性
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> ◊
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> ◊ 柱高与分辨率（选择性）
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> ◊
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> ◊ 运行流速与峰宽（有效性）-影响因素
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> ◊
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> 运行流速与峰宽（有效性）-层析速率方程
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> ◊
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> ◊
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> 运行流速与峰宽（有效性）-实例
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> ◊
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> ◊ 填料的粒径与选择性
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> ◊ 填料的装填柱效与有效性
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> ◊
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> ◊ 上样体积和上样浓度
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> ◊
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> ◊ 样品和缓冲液的粘度
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> ◊ 缓冲液的成分
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◊
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> ◊ 层析柱接线管路的长度和直径
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◊
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- 主要作用
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◊ 蛋白精纯、样品筛选鉴定、疫苗纯化和质控、样品换液
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- 常见凝胶过滤应用及填料选择
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◊ S 系
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> ◊
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> ◊ Sephadex G系列
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> ◊
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> ◊ Superdex系列
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> ◊
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> ◊
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> ◊
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> ◊
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> ◊ Superose系列
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> ◊
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> ◊ Sepharose系列
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> ◊
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> ◊
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> ◊
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> ◊ 1
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> ◊ Sephacryl系列
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> ◊
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> ◊
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> ◊
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- 层析柱的使用
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◊ 凝胶过滤层析柱的运行参数及填料数据
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◊ 可找供应商要
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◊ 凝胶过滤层析柱的装填
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◊ 两部流速填装（不要超压）
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> 
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- 层析柱的反向清洗
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◊
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多次使用后填料会变脏，反压会变大
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> ◊
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> ◊ 一般10次\~20次进行一次反向清洗（柱子下端较上端洁净）
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- 层析柱的保存
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> ◊
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> ◊ 1
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- 层析柱滤膜的更换
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◊ 如果在清洗后层析柱依然很脏、反压很大则可以更换上端的滤膜
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> ◊
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