GE离子交换层析.pdf

离子交换层析一种广泛应用于各种纯化阶段和规模的层析技术什么是离子交换层析?离子交换层析是一种根据被分离物质的分子表面电荷性质来分离的吸附层析通过电荷性质分离相反电荷性质之间的作用 蛋白质上的电荷与填料表面的电荷相互作用，不同的蛋白质带有电荷性质不同，与填料的作用也就不同 阴离子交换和阳离子交换 蛋白质带有负电荷时叫做阴离子交换 蛋白质带有正电荷时叫做阳离子交换Some of the charged regions which will influence ion exchangeDifferent proteins have different charges and different patterns of surface charge选择性的原理NH3RCOOH+NH3RCOO+-RNH2COO-low pHpositive chargehigh pHnegative chargehydrogen gainedhydrogen lostpH 对电荷性质的影响overall charge on protein-+NH3RCOOH+NH3RCOO+-RNH2COO-acid isoelectric pointalkalineexcess positive charge balanced positive and negative charge excess negative charge表面电荷的性质和多少依赖于 pHpH 3pH 10滴定曲线pH 3pH 10anion exchangercation exchangercharge on protein-+通过 pH控制选择性+-平衡离子交换过程阴离子交换介质-+-上样和清洗离子交换过程-+-洗脱离子交换过程-+再生离子交换过程带电荷基团阴离子交换基团Diethylaminoethyl(DEAE)-OCH2CH2N+H(CH2CH3)2Quaternary aminoethyl(QAE)-OCH2CH2N+(C2H5)2CH2CHOHCH3Quaternary ammonium(Q)-CH2N+(CH3)3阳离子交换基团Carboxymethyl(CM)-OCH2COOSulphopropyl(SP)-CH2CH2CH2SO3Methylsulphonate(S)-CH2SO3weak ion exchangers:capacity varies with pH weak anionexchangerweak cationexchangerstrong anion exchangerstrong cation exchangerstrong ion exchangers:capacity is constant over a wide range of pHQ SepharoseFast FlowSP Sepharose Fast FlowDEAE Sepharose Fast FlowCM Sepharose Fast Flow强离子交换和弱离子交换为什么选择离子交换层析?可用于各个纯化阶段和各种规模 可控性 高选择性 高载量 浓缩的 高回收率Results can be optimised via several factorsEffect of pH on fractionation of model proteins良好的可操控性考虑因素 需要什么样的设备?需要什么样的离子交换?怎么操作分离过程?pHUVcondfraction collection Frac-901AMIXERW1W2Bsamplepump P-920valve FV-903monitor UPC-900different buffersinjection valve V1large and small fractionscolumns for AIX,CIX and buffer exchangemonitor for UV,conductivity and pH离子交换的通用装备交换基团 类型 密度基质 多孔性 粒度大小 化学性质 选择性 载量 载量,流速 峰宽,流速 使用寿命可再生性离子交换填料HiTrap IEX Selection Kit:conalbumin(I),a-lactoglobulin(II)and soya bean trypsin inhibitor(III)Sample:0.4 mg conalbumin pI=6.3,0.8 mg-lactoglobulin pI=5.8,1.2 mg soya bean trypsin inhibitor pI=4.5 dissolved in 2 ml start buffer.Columns:HiTrapDEAE SepharoseFast Flow 1 mlHiTrap Q Sepharose Fast Flow 1 mlHiTrap Q XL 1 mlHiTrap ANX Sepharose 4 Fast Flow high sub 1 mlStart buffer:20 mM Tris-HCl pH 7.4Elution buffer:20 mM Tris-HCl,0.5 M NaCl pH 7.4Flow rate:1 ml/min 150 cm/hRunning parametersequilibration:20 ml start buffersample application:2 mlwash:5 ml start buffer elution 40 ml,linear gradient,080%elution bufferInstrumentation:KTAexplorer100 controlled by UNICORN3.0使用不同的填料优化层析效果强离子交换的优势 在很宽的 pH范围下都带有电荷 在很宽的 pH范围下电荷密度不变 比较快速容易地达到平衡多孔性与载量Ion exchangerPorosityMax load/mlMini Qsolidca 5 mgMono Qvery highca 50 mg机械强度好才能高流速X 大颗粒允许较大的体积流速填料和流速MiniBeads3 mmicro-purificationMonoBeads10 mpolishingSOURCE1515 mpolishingSOURCE 3030 mintermediate steps颗粒大小3 m Mini S10 m Mono S填料颗粒大小与分辨率载量类型总离子载量e.g.140-200 mol/mL并不重要有效载量e.g.25 mg HSA/mL随着pH、样品、离子强度不同而变化动态载量e.g.25 mg HSA/mL,300 cm/h与流速有关也随着pH、样品、离子强度不同而变化现代填料的要求 良好的机械强度，流速快 高载量 良好的可再生能力 不同的操作阶段使用不同颗粒大小的填料合适 的层析条件 pH 添加剂 洗脱方式 流速在几个试管中各放 1 ml 离子交换填料加入不同 pH值的缓冲液加入样品并摇匀分析上清中目的蛋白的情况左图中目标蛋白在pH7.5时完全吸附结论:阴离子交换,结合缓冲液选 pH 7.56.06.57.07.58.0pH试管检测法确定结合缓冲液的 pH滴定曲线实验方法滴定曲线结果elution volume(ml)01020304050020406080100120mAUM NaCl10.50pH 5pH 6pH 7pH 8pH 9通过 scouting优化pHColumn:RESOURCEQ,6 mlSample:2 mg crude pancreatinSystem:KTAexplorer100,BufferPreppH 0.5pH 0.5A.Citrate 20 mM,pH 4.9B.Citrate 20 mM,NaCl 1 M,pH 4.9A.Citrate 20 mM,pH 4.9 B.A+NaCl 1 M 不同的方法来配制的“相同的”缓冲液To ensure a proper pH value,the sample should be dissolved in start buffer(A),critical with large volume samplesBuffering ion concentrations of 2050 mM are usually sufficientAUvolumepH valuegradientvolumeAUpH valuegradientpH 值和缓冲能力谢谢欢迎访问: Q,Mini S,MonoBeads,Mono Q,Mono S,PhastGel,PhastSystem,RESOURCE,Sephadex,Sepharose,SMART,SOURCE,STREAMLINE,Tricorn,UNICORN,KTAexplorer and KTApilot are trademarks of GE Healthcare Ltd,a General Electric Company.GE,imagination at work and GE Monogram are trademarks of General Electric Company.Coomassie is a trademark of ICl plc.All goods and services are sold subject to the terms and conditions of sale of the company within GE Healthcare which supplies them.General Electric Company reserves the right,subject to any regulatory and contractual approval,if required,to make changes in specifications and features shown herein,or discontinue the product described at any time without notice or obligation.2006 General Electric Company All rights reserved.GE Healthcare Bio-Sciences AB,a General Electric Company.GE Healthcare Bio-Sciences AB,Bjrkgatan 30,SE-751 84 Uppsala,Sweden.28-4085-42