| 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.PrepareKLH-HaptenConjugation:
1.1 Add200µLofddH2OintothevialofmcKLH(ComponentA)tomakea10mg/mLsolution.
Note:mcKLHsolutionappearstranslucenttowhitish-bluetypically.Donotvortexorheatthesolution,whichwillprecipitatethecarrier.
1.2 Dissolveupto2mghaptenin450mLConjugationBuffer(ComponentB).
Note:Somehaptensmighthavelimitedsolubility,useDMSO(<30%inthefinalconjugationsolution)todissolveitfirst.HigherconcentrationofDMSOmightirreversiblydenaturethecarrierprotein.
1.3 Addthe450mLhaptensolution(fromStep1.2)intothe200µLofmcKLHsolution(fromStep1.1)tohaveKLH-Haptenmixturesolution.
1.4 DissolveonevialofEDC(ComponentC,10mg)in1mlofddH2Oandimmediatelyadd50µlofthissolutiontotheKLH-Haptensolution(fromStep1.3),mixitgently.Incubateatroomtemperaturefor2hours.
1.5 Purifytheconjugatebydesaltingtoremovenon-reactedcrosslinkerandproteinpreservative(e.g.,sodiumazide).
2.PurifyKLH-Haptenconjugate:
2.1 Twistoffthebottomclosureofthedesaltingcolumn(ComponentE),andloosenthecap.Placethecolumninacollectiontube.
2.2 Centrifugethecolumnat1,000gfor2minutestoremovethestoragesolution.
2.3 Removethecapandslowlyadd1mLofpurificationbuffertothecolumn.Centrifugeat1,000gfor2minutes,removethebuffer. Repeatthisstepfor3additionaltimes,discardingthebufferfromthecollectiontube.
2.4 Placethecolumntoanewcollectiontube,andgentlyapplythesampleintothecenterofthecompactresinbed.
2.5 Centrifugethecolumnat1,000gfor2minutestocollectthesample.
2.6 TheKLH-Haptenconjugatecannowbeusedforimmunization.IftheKLH-Haptenconjugateistobestoredformorethanafewdays,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 |
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