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基质为疏水
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样品处理原理为盐析，破环水化膜，暴露疏水核，才能与疏水介质进行结合
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经验：定量蛋白，不断加硫酸铵，直至要盐析的状态：泛白，搅拌溶解
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洗脱：第1个峰：分离峰，冲杂蛋白，冲洗体积大
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0.35峰：过渡峰，部分收集
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0.05峰：收集峰、富集峰，目的蛋白的主要收集
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> Phenyl
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- 目的
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□
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> 捕获目的蛋白，除去宿主蛋白（相关蛋白质）
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- 层析原理
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□ 疏水作用，盐析作用，通过控制离子强度，高盐上样，低盐洗脱
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- 层析柱型号
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柱内直径（cm）
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柱高（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mg/ml
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蛋白处理范围（g）
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INDEX 140/500
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14
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50
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5.5±0.5
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35±5
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20\~35
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100\~210
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> ○
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- 填料信息
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- 名称：Phenyl Sepharose 6 Fast Flow（high sub）
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- 更换时间
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- 柱效要求
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2014年
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2021.07
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> □ 每米理论塔板数：≥2000
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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
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- 0.7mol/L (NH4)2SO4-10m mol/L Tris-HCl-1m mol/L EDTA
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- 样品调节缓冲液
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- 3.5mol/L (NH4)2SO4溶液、1mol/L Tris-HCl-100mmol/L EDTA
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- 冲洗缓冲液
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- 0.7mol/L (NH4)2SO4-10m mol/L Tris-HCl-1m mol/L EDTA
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- 洗脱液
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- 备注
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- 0.5mol/L (NH4)2SO4-10m mol/L Tris-HCl-1m mol/L EDTA
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- 0.35mol/L (NH4)2SO4-10m mol/L Tris-HCl-1m mol/L EDTA
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- 0.05mol/L (NH4)2SO4-10m mol/L Tris-HCl-1m mol/L EDTA
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- 能较好反应前端工序情况
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- 收集峰形可能变化较大
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- Q&A
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Phenyl工序对上样液调样时，不考虑里边有0.1mol/L氯化钠，直接规定了硫酸铵浓度为0.7mol/L，而不是规定一个电导率范围
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Phenyl工序调样容易产生絮状物，容易堵柱子和增大反压，手法应该怎么改进，减少絮状物的产生
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Phenyl工序上样后阶段性出现多次柱头接触的胶出现裂缝的现象，但为什么对其收率及相关蛋白质影响不是很大调样后上样液的电导率要求是11.5±0.5mS/cm，是否与平衡液电导率一致或者高一点吸附效果更好
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如果上样过程中出现干柱现象，应该怎么处理
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Phenyl工序使用不同浓度的硫酸铵洗脱蛋白，目的蛋白在0.35\~0.05mol/L之间洗脱，分别为3、4、5\#样，为什么不直接使用
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0.05mol/L的直接洗脱收集3、4、5\#样
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> 分区 层析 的第 1 页
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