Duringthepastfivedecades,varioustypesofchemistrieshavebeenusedforconjugationofmoleculessuchasantibodiestothesurfaceoftheliposomes.Ingeneral,theconjugationcanbeachievedthroughtheN-terminus,theC-terminusortheavailablesulfur(e.g.Fab’fractionorthiolatedAb).NotallchemistrieshavethesameyieldandefficiencyofconjugationandoftenreproducingbiocompatIBLebatchescanbeachallenge.Theliposomescontainingpyridyldithiopropionate(PDP)lipidsareusedtoconjugateproteins,antibodiesandothermoleculescontainingthereactivemoiety.PDPlipidsarenotaswidelyusedasmaleimidelipids,buttheydohavetheirownnicheapplication.ThePDPgroupcontainsdisulfide,whichcanreactwithsulfhydrylorthiolatedproteins/antibodies.Therefore,PDP-functionalizedliposomescanbeusedintwoways:
MethodA.Inthisapproach,thepyridyldithiogrouponthedistalendsofthePEGchainscontainsPDPisforestreducedbyareducingagent(dithiothreitol,DTT).Maleimide-containingantibodiesarethenefficientlycoupledtothesurfaceofliposomes.Thethiol-maleimideprocedureisoneofthemostdesirablereactionsinbioconjugatechemistryduetoitssimplicityandhighcouplingefficiencyinaqueoussolution.Thereaction,whichisbasedonthestablethioetherlinkagebetweenathiolgroup(reducedformofPDP-liposome)andthecorrespondingmaleimidegroup,occursselectivelyandirreversiblyatneutralpH(6.5-7.5),andtheformedbondsarenotcleavedbyreducingagents.Inaddition,duetothepresenceoftwodifferentoxidationstatesofsulfurresidues(oxidizedandreducedstatesasadisulfidebondandsulfhydrylgroup,respectively)onthetwoconjugatingcomponents(i.e.,theliposomeandprotein/antibody),theprobABIlityofthecrosslinkingofthehomologousagentsislow.Therefore,protein-proteinandliposome-liposomecrosslinkingdoesnotusuallyhappen.
MethodB.Alternatively,thePDPgroupcanparticipateindisulfideexchangereactionswiththiolspresentontargetingproteins/antibodies.Thecouplingreactionisfastandconductedundermildconditions.However,theformeddisulfidebondshavebeenreportedtobelessstablethanthioetherbonds.Moreover,eveninanalkalinemedium(pH8.0),thiolgroupsareoxidized.Thedisulfidebondformedbetweentheprotein/antibodyandliposomescanalsobebrokeninthepresenceofareducingagentandtherefore,theconjugationreactionisreversible.
MethodA. Conjugationofmaleimide-modifiedantibodytoaPDP-modifiedliposome.
MethodB.Conjugationofathiol-modifiedantibodytoaPDP-modifiedliposome.
ImmunoFluor™-PDPisaPEGylatedproduct.Forotherreactive(PEGylatedandnon-PEGyalatedproducts)ImmunosFluor™productssuitableforothertypesofconjugationmethodsee here.
ebiomall.com
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试剂盒里有详细的说明书,告诉你样品需要多少量,每个试剂需要加入多少量,和详细的实验步骤,一般买来就可以用,不用人教。
所以你问一个样需要多少量是没法回答的,测定过程是要加很多种试剂的。
谢谢谢
现在跑tricine-sds-page电泳老是跑不好,
我刚做了bradford定量,
觉得差异与空菌对照还是挺明显的,
但是这个方法只能初略的估计,
还是不能精确定量,
做HPLC我现在还没有条件,
所以我想能不能用BCA或者lowry法先进行定量啊?
这种方法如果做出来结果的话,
能不能发个sci之类的文章啊?
希望高手们指点一下!
谢谢了
LOQ-定量限LOD-检测限
至少可以说同一种蛋白定量试剂盒并不适用于所有细胞
蛋白定量试剂盒有分Bradford,BCA,Lowry等,每一种对不同的细胞都有独特的适用范围和灵敏度。所以需要多种蛋白定量试剂盒对比检测才能让结果准确
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