蛋白质的分离与纯课件.pptVIP

蛋白质的分离与纯化;Selectionofproteinsource蛋白质原料的选择;Assayofproteins蛋白质的检测;溶液中加入一定量的中性盐如[NH4]2SO4

A由于[NH4]2SO4的亲水性比蛋白质亲水性大，能与大量的水分子结合，使蛋白质表面水膜退化、消失；

B[NH4]2SO4解离，中和了蛋白质颗粒所带的电荷，破坏蛋白质表面电荷层而沉淀析出;（二）有机溶剂沉淀蛋白质：

凡能与水以任意比例混合的有机溶剂，如乙醇、甲醇、丙酮等，均可用于沉淀蛋白质。

沉淀原理是：①脱水作用；②使水的介电常数降低，蛋白质溶解度降低。

为了防止蛋白质在分离过程中发生变性，有机溶剂浓度不能太高(30%-50%)，而且需要在低温条件下进行。;（三）层析：;Gelfiltrationchromatography

凝胶过滤层析（1）;Gelfiltrationchromatography

凝胶过滤层析（2）;Gelfiltrationchromatography凝胶过滤层析（4）;Ionexchangechromatography

离子交换层析（2）;Ionexchangechromatography离子交换层析（3）;Ion-exchangechromatographyexploitsdifferencesinthesignandmagnitudeofthenetelectricchargesofproteinsatagivenpH.Thecolumnmatrixisasyntheticpolymercontainingboundchargedgroups;thosewithboundanionicgroupsarecalledcationexchangers,andthosewithboundcationicgroupsarecalledanionexchangers.Ion-exchangechromatographyonacationexchangerisshownhere.TheaffinityofeachproteinforthechargedgroupsonthecolumnisaffectedbythepH(whichdeterminestheionizationstateofthemolecule)andtheconcentrationofcompetingfreesaltionsinthesurroundingsolution.SeparationcanbeoptimizedbygraduallychangingthepHand/orsaltconcentrationofthemobilephasesoastocreateapHorsaltgradient.;Ionexchangechromatography

离子交换层析（4）;Affinitychromatography

亲和层析（1）;Affinitychromatography亲和层析（2）;(c)Affinitychromatography

separatesproteinsbytheirbindingspecificities.

Theproteinsretainedonthecolumnarethosethatbindspecificallytoaligandcross-linkedtothebeads.(Inbiochemistry,theterm“ligand”isusedtorefertoagroupormoleculethatbindstoamacromoleculesuchasaprotein.)Afterproteinsthatdonotbindtotheligandarewashedthroughthecolumn,theboundproteinofparticularinterestiseluted(washedoutofthecolumn)byasolutioncontainingfreeligand.;AmodernrefinementinchromatographicmethodsisHPLC,orhigh-performanceliq