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非全排阻
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大分子难进，难深入
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除二聚体、四聚体
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比全排阻更注重介质完整性
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> S-100
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> 2021年6月24日 21:49
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- 盐析（去除硫酸铵、Tris，缩小体积，将缓冲体系更换为PB）
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- 盐析
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□
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□
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□
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□
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> 目的：使蛋白沉淀出来温度：？
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> 硫酸铵用量：3.5mol/L硫酸铵溶液→硫酸铵终浓度：1.35mol/L～1.83mol/L
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> 时长：10min
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- 一次离心
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- 目的：使蛋白沉淀与杂质溶液分离
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□ 温度：4℃
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□ 离心力：9910g
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- 时长：30min
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- 留存：蛋白沉淀
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- 蛋白溶解
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- 目的：使蛋白沉淀溶解
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- 溶解液：5m mol/L PB pH 7.2±0.1
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- 蛋白浓度：35mg/ml\~50mg/ml
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- 二次离心
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□
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□
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□
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- S-100
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- 目的
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□
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> 目的：使目的蛋白溶液与杂质蛋白沉淀分离
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> 温度：4℃
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> 离心力：9910g 时 长 ：15min 留存：上清液
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> 除去高分子蛋白质
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- 层析原理
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- 分子筛，非全排阻，每米理论塔板数很重要
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- 层析柱型号
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柱内直径（cm）
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柱高度（cm）
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柱床体积（L）
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柱床高度（cm）
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上样量
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蛋白处理范围
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BPG 200/950
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20
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95
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25.0±0.5
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80±5
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≤6%柱床体积
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≤1.47L
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> ○
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- 填料信息
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- 名称：Sephacryl S100 H.R
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- 排阻范围：1\~100KD
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- 更换时间
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- 柱效要求
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- 每米理论塔板数：≥4000
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□ 对称性：0.8\~1.2
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- 缓冲液
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- 平衡缓冲液、洗脱缓冲液
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- 0.05mol/L NaCl-5m mol/L PB
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- 步骤
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- 平衡
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- 上样
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- 洗脱
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- 收集
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- Q&A
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- S-100浓度多少开始收集，浓度多少可以结束，除了浓度收集法，还有什么收集方法？
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- SOP中规定，S-100工序后处理后需要用WFI冲至中性，但因生产计划3天1批导致没有时间冲到中性，然后继续下一批生
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产，是否合规
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