2017_protein_purification(1).pptVIP免费

1Proteinpurification2017-10-10chunguangwang@tongji.edu.cn王春光234BasicprincipleofproteinpurificationTheuniquepropertymatters!5ProteinsizeNH2-CH-COOH│CH2COOHMr=133aminoacid-NH-CH-CO-│CH2COOHMr=115residueAveragemolecularweightofresidues=110.Example:aproteinof22KDabout200residues;cDNAofabout600bp.6Gelfiltrationchromatography凝胶过滤层析Alsoknownassizeexclusionchromatography;Effectiveseparationrange(globularproteins);Exclusionlimit;Limitedsamplevolume;Bedvolumeandvoidvolume;Scanningelectronmicrographofanagarosegel.Magnificationx50,000.(Ref.AndersS.Medin,PhDThesis,UppsalaUniversity1995)789Example:Superdex75fromGEHealthcare10Ionexchangechromatography(fromGEHealthcare)11Ionexchangechromatography离子交换层析1213TypeofIonExchangeCommonAbbreviationFunctionalgroupStrongAnionQQuarternaryAmmonium─CH2N+(CH3)3WeakAnionDEAEDiethylaminoethyl─C2H4N+H(C2H5)2StrongCationSSulfonicAcid─CH2SO3-WeakCationCMCarboxymethyl─CH2COO-Ionexchangechromatography14IonexchangechromatographyCouldbeelutedbyincreasingsaltconcentrationorchangingpHcondition;Stepwiseelution（阶段洗脱）orgradientelution（梯度洗脱）;010203040500.00.20.40.60.81.0[Salt](M)Elution(ml)010203040500.00.20.40.60.81.0[Salt](M)Elution(ml)15Example:MonoScolumnfromGEHealthcareRibonucleaseApI=9.45CytochromeCpI=10.0-10.5LysozymepI=11.3516Hydrophobicinteractionchromatography(HIC)（疏水相互作用层析）Hydrophobicity:octyl>phenyl>butyl(http://www.chisso.co.jp/fine/en/cellufine/images/hyd_partial.gif)(fromGEHealthcare)17Affinitychromatography（亲和层析）Flash18Immobilizedmetalchelatingchromatography(IMAC)Popularlyusedfor(His)6-taggedproteins.Imidazoletoelute;Moreoptionsofsomeothermetalions(Co2+,Cu2+);(fromQiagen)19proteinpurificationtechniquesBasedonphysicalandchemicalcharacteristics:sizechargegelfiltrationchromatographyhydrophobicityBasedonbiologicalcharacteristics:affinitychromatographyionexchangechromatographyhydrophobicinteractionchromatography20ChangeconditiontoeluteNoYesYesYesSamplevolumelimitedYesNoNoNo21AKTApurifier10fromGEHealthcare22AKTApurifier10fromGEHealthcare