Overview | PrinterFriendlyVersion |
Ex/Em(nm) | None/None |
MW | N/A |
CAS# | N/A |
Solvent | N/A |
Storage | R |
Category | ProteinBiochemistry Generalproteins |
Related | BiochemicalAssays |
Thefollowingprotocolisageneralprotocolforawidevarietyofhaptens.Optimizetheprotocolaccordinglyfortheconjugationefficienciesuponthesizeandstructureofyourhapten.Usingamolarexcessofhaptenovercarrierproteinensuresefficientconjugation.Ingeneral,areactionwithequalmassamountsofhaptenandcarrierproteinwillachievesufficientmolarexcess.
1.PrepareBSA-HeptenConjugation:
1.1 Add200µLofddH2OintothevialofBSA(ComponentA)tomakea10mg/mLsolution.
1.2 Dissolveupto2mghaptenin450mLConjugationBuffer(ComponentB).
Note:Somehaptensmighthavelimitedsolubility,useDMSO(<30%inthefinalconjugationsolution)todissolveitfirst.HigherconcentrationofDMSOmightirreversiblydenaturethecarrierprotein.
1.3 Addthe450mLhaptensolution(fromStep1.2)intothe200µLBSAsolution(fromStep1.1)tohaveHapten-BSAmixturesolution.
1.4 AddtheHapten-BSAsolution(fromStep1.3)intoonevialofEDC(10mg),dissolveitbygentlemixing.Incubateatroomtemperaturefor2hours.
1.5 Purifytheconjugatebydesaltingtoremovenon-reactedcrosslinkerandproteinpreservative(e.g.,sodiumazide).
2.PurifyBSA-Heptenconjugate:
2.1 Thaw1bottleofPurificationBuffer(ComponentD)toroomtemperaturebeforeuse.Unusedbuffercanbestoredat4°Cfor1week.
2.2 Twistoffthebottomclosureofthedesaltingcolumn(ComponentE),andloosenthecap.Placethecolumninacollectiontube.
2.3 Centrifugethecolumnat1,000gfor2minutestoremovethestoragesolution.
2.4 Removethecapandslowlyadd1mLofpurificationbuffertothecolumn.Centrifugeat1,000gfor2minutes,removethebuffer. Repeatthisstepfor3additionaltimes,discardingthebufferfromthecollectiontube.
2.5 Placethecolumntoanewcollectiontube,andgentlyapplythesampleintothecenterofthecompactresinbed.
2.6 Centrifugethecolumnat1,000gfor2minutestocollectthesample.
2.7 TheHapten-BSAconjugatecannowbeusedforimmunization.IftheHapten-BSAconjugateistobestoredformorethanafewdays,sterilefiltertheconjugate,andstoreat4°Cor-20°C.
Note1:Iftheconjugateistobeusedwithinoneweek,PBSmaybeusedforpurification.Iftheconjugatewillbefrozen,usethepurificationbuffersalts(ComponentD)forpurification.
Note2:IfDMSOisusedintheconjugation,preparethepurificationbuffersaltswiththesamepercentageofDMSOusedforconjugation.Thiswillminimizetheprecipitationinthecolumnduringdesalting.
Note3:Ifaprecipitateformedduringconjugation,centrifugetheprecipitatedmaterial,collectthesupernatantandsavetheprecipitate.Purifythesupernatant.Combinetheprecipitateandthepurifiedconjugate.
References&Citations | PrinterFriendlyVersion |
1. ZuY,ZhangY,ZhaoX,ZhangQ,LiuY,JiangR.(2009)Optimizationofthepreparationprocessofvinblastinesulfate(VBLS)-loadedfolate-conjugatedbovineserumalbumin(BSA)nanoparticlesfortumor-targeteddrugdeliveryusingresponsesurfacemethodology(RSM).IntJNanomedicine,4,321.
2. EnomotoH,LiCP,MorizaneK,IbrahimHR,SugimotoY,OhkiS,OhtomoH,AokiT.(2008)Improvementoffunctionalpropertiesofbovineserumalbuminthroughphosphorylationbydry-heatinginthepresenceofpyrophosphate.JFoodSci,73,C84.
3. Ledesma-OsunaAI,Ramos-ClamontG,Vazquez-MorenoL.(2008)Characterizationofbovineserumalbuminglycatedwithglucose,galactoseandlactose.ActaBiochimPol,55,491.
4. WangJH,WangHQ,ZhangHL,LiXQ,HuaXF,CaoYC,HuangZL,ZhaoYD.(2007)PurificationofdenaturedbovineserumalbumincoatedCdTequantumdotsforsensitivedetectionofsilver(I)ions.AnalBioanalChem,388,969.
5. ZhengC,BiJ,MaG,SuZ.(2007)Polyethyleneglycolimprovesconjugationofbovinehemoglobinandhumanserumalbumininacontrolledratio.ArtifCellsBloodSubstitImmobilBiotechnol,35,568.
6. TattiniV,Jr.,ParraDF,PolakiewiczB,PitomboRN.(2005)EffectoflyophilizationonthestructureandphasechangesofPEGylated-bovineserumalbumin.IntJPharm,304,124.
7. BurkinAA,KononenkoGP,SobolevaNA.(2003)[Productsofspontaneousconjugationofaflatoxinswithbovineserumalbumin:immunochemicalproperties].PriklBiokhimMikrobiol,39,228.
8. MaoSJ,HouSX,ZhangLK,JinH,BiYQ,JiangB.(2003)[Preparationofbovineserumalbuminnanoparticlessurface-modifiedwithglycyrrhizin].YaoXueXueBao,38,787.
9. ConlanJW,ShenH,WebbA,PerryMB.(2002)MicevaccinatedwiththeO-antigenofFrancisellatularensisLVSlipopolysaccharideconjugatedtobovineserumalbumindevelopvaryingdegreesofprotectiveimmunityagainstsystemicoraerosolchallengewithvirulenttypeAandtypeBstrainsofthepathogen.Vaccine,20,3465.
10. NakamuraM,ObaY,MoriT,SatoK,IshidaY,MatsudaT,NakamuraH.(2002)Generationofpolyclonalantibodyagainstmu-conotoxinGIIIAusinganimmunogenof[Cys(5)]mu-conotoxinGIIIAsite-specificallyconjugatedwithbovineserumalbumin.BiochemBiophysResCommun,290,1037.