蛋白纯化策略VIP免费

1/GE/PurificationStrategies纯化策略Introduction介绍BeforePurification…纯化前的准备Strategies纯化策略PurificationofrecombinantandnativeProtein纯化实例Howtogetdesiredresolution?如何取得预期的结果？Summary总结ContentGeneratedbyFoxitPDFCreator©FoxitSoftwarehttp://www.foxitsoftware.comForevaluationonly.2/GE/Introduction介绍BeforePurification…纯化前的准备Strategies纯化策略PurificationofrecombinantandnativeProtein纯化实例Howtogetdesiredresolution?如何取得预期的结果？Summary总结Content4/GE/2010-4-13Principlesofoperationforchromatographytechniques层析原理GelFiltration分子筛IonExchange离子交换Hydrophobicinteraction疏水层析Affinity亲和Reversedphase反相SOOOGeneratedbyFoxitPDFCreator©FoxitSoftwarehttp://www.foxitsoftware.comForevaluationonly.3/GE/5/GE/2010-4-13HowtousetheseseparationtechnologiestoperformaSUCCESSFULpurification?如何运用各种分离纯化技术来实现成功的分离纯化？QuestionIntroduction介绍BeforePurification…纯化前的准备Strategies纯化策略PurificationofrecombinantandnativeProtein纯化实例Howtogetdesiredresolution?如何取得预期的结果？Summary总结ContentGeneratedbyFoxitPDFCreator©FoxitSoftwarehttp://www.foxitsoftware.comForevaluationonly.4/GE/7/GE/2010-4-13目标:开发有效的蛋白质分离纯化工艺•Maintainedbiologicalactivity活性•Sufficientpurityandquantity纯度&规模•Goodeconomy经济性8/GE/2010-4-13pgngµgmggkg治疗用蛋白结构研究功能研究质谱设定目标：需要的蛋白量用于免疫的抗原GeneratedbyFoxitPDFCreator©FoxitSoftwarehttp://www.foxitsoftware.comForevaluationonly.5/GE/9/GE/2010-4-13Extremelyhigh极高High高Moderate中等•therapy治疗用•invivostudiesPK/PD动物实验•crystallizationforx-raystudies结晶•N-terminalsequencingofanunknownproteinN端测序•mostphysical-chemicalcharacterizationmethods鉴定•antigenformonoclonalantibodyproduction免疫终产品纯度要求–一般原则:10/GE/2010-4-13选择不同规模、不同功能的系统ÄKTAprime™plusÄKTAexplorer™ÄKTAprocess™ÄKTApilot™ÄKTAxpress™LABSCALEBIOPROCESSSCALESIMPLEPURIFICATIONADVANCEDPURIFICATIONINCREASEDTHROUGHPUTLABSCALELOWTHROUGHPUTÄKTApurifier™GeneratedbyFoxitPDFCreator©FoxitSoftwarehttp://www.foxitsoftware.comForevaluationonly.95%及以上6/GE/11/GE/2010-4-13开始纯化之前样品来源•原核or真核?•胞内or胞外?细胞破碎?•活性,复性?目标蛋白的性质•等电点•分子量•稳定性(pH/salt,etc)……InnermembranePeptidoglycanOutermembraneLipopolysaccharide70Å70Å70Å210Å胞内~2000proteins~100proteins细胞间质~10proteins胞外12/GE/2010-4-13Targetproteinstabilitywindow目标蛋白的稳定性DeterminationofasuitableammoniumsulfateconcentrationandpHscreeningrangeforHICGeneratedbyFoxitPDFCreator©FoxitSoftwarehttp://www.foxitsoftware.comForevaluationonly.microcal差示扫描量热仪7/GE/13/GE/2010-4-13开始纯化之前建立目标蛋白特异性检测方法•ActivityAssay(specific)快速可靠的活性检测方法Arapidandreliableassayforthetargetprotein•Puritydetermination纯度测定方法–e.g.SDS-PAGE电泳,HPLC高效液相层析•Totalproteindetermination总蛋白测定–e.g.colorimetricmethod比色法主要杂质的性质及检测方法AgIntroduction介绍BeforePurification…纯化前的准备Strategies纯化策略PurificationofrecombinantandnativeProtein纯化实例Howtogetdesiredresolution?如何取得预期的结果？Summary总结ContentGeneratedbyFoxitPDFCreator©FoxitSoftwarehttp://www.foxitsoftware.comForevaluationonly.8/GE/15/GE/2010-4-13策略1：纯化三步曲(CIPP)Threephasestrategypuritystepcaptureintermediatepurificationpolishingisolateproduct,concentrate,stabilizer...