BAND-SHIFTTheproceduredescribedinthischapterforthedeterminationofaffinityconstantsandkineticdissociationconstantsbyband-shiftassayreferstoanidealantibodyfragment(e.g.,ascFvoranFabfragment)bindingtoawell-behavedproteinantigen(pure,ofwell-definedoligomericstate,migratingasasinglebandinnon-denaturinggelelectrophoresis.Asmentionedintheintroduction,eithertheantibodyortheantigenhastobelabeledintheseassays.Inordertoillustratedifferentlabelingmethods,wewilluserADIolabeledrecombinantantibodiesintheprotocolforthedeterminationofKd,andfluorescentlylabeledantigenintheprotocolforthedeterminationofk_off.Recombinantantibodiescanconvenientlyberadiolabeledsite-specificallyandwithoutlossofimmunoreactivityusinggeneticallyintroducedphosphorylationsites.ThisprocedureisdescribedinthisChapter.However,otherradiolabelingmethodscanbeused(e.g.,radioiodinationusingIodogentubes),providedthatimmunoreactivityispreservedandthatasufficientlyhighspecificactivityisachieved.ThefluorescentantigenlabelingmethodpresentedinthisChapterfeaturestheuseofN—hydroxysuccinimidoesterderivativesofacommerciallyavailablecyaninedye(Cy5—NHS;AmershamPharmaciaBiotech).Thesereagentsreactwithprimaryaminogroupsintheantigenmolecule,andmayimpairtheantigen’sABIlitytoreactwiththeantibodyafterlabeling.Phosphorylationofrecombinantantibodies

DissolveinbufferAthepurifiedantibodycarryinganengineeredphosphorylationsite(e.g.,thesequenceDDDSDDDD;at1mg/mlconcentration(alternatively,exchangebufferbydialysis).

To100µlantibodysolution,add500unitscaseinkinaseII(4µl;NewEnglandBiolabs)and1.6mCiof[g-³²P]ATP(12µl,Cat.No.35020fromICNBiomedicals).

Incubateatroomtemperaturefor1hour.

RemoveunreactedATPusingadisposablePD-10column(AmershamPharmaciaBiotech),preblockedwithbovineserumalbumindissolvedinPBS(2mg/ml)andextensivelywashedwithPBS.Thisgelfiltrationprocedureisperformedbyloadingthe116µlreactionmixtureontothecolumn,waitinguntilthecolumnisdry,adding2.4mlPBS(withoutcollectingtheliquid),waitinguntilthecolumnisdry,andfinallyadding2mlofPBS,collectingtheliquidfromthecolumn.Thisfractionofradiolabeledantibodycanbeusedinband-shiftassays.

Labelingtheantigenwitharedfluorophore

DissolveinPBSthepurifiedantigenat1mg/mlconcentration(alternatively,exchangebufferbydialysis).

Add1mltoadrytubecontaininganadequateamountofCy5-NHSforthelabelingof1mgprotein(Cat.No.PA25001—monoreactiveCy5dyepackfromAmershamPharmaciaBiotech).

Incubate30min.atroomtemperature.

RemoveunreactedCy5dyeusingadisposablePD-10column(AmershamPharmaciaBiotech),extensivelypre-washedwithPBS(>25ml).Thisgelfiltrationprocedureisperformedbyloadingthe1mlreactionmixtureontothecolumn,waitinguntilthecolumnisdry,adding1.5mlPBS(withoutcollectingtheliquid),waitinguntilthecolumnisdry,andfinallyadding2.5mlofPBS,collectingtheliquidfromthecolumn.AclearseparationbetweenincorporatedandunincorporateddyeshouldbevisIBLe.Thecollectedblue-colouredfractionoffluorescentlylabeledantigencanbeusedinband-shiftassays.

Nativegelelectrophoresis

Proteinsamples(4µl)aredilutedwith2µlofgelmiximmediatelybeforetheelectrophoreticseparation.

ApplyproteinsamplesontoaGradient8-25PhastGel(AmershamPharmaciaBiotech;adifferentgelpercentagemayberequired,dependingonthemobilityoftheproteinswhichhavetobemeasured),equippedwithnativebufferstripsinaPhastSystemseparationmodule(AmershamPharmaciaBiotech)andrunthegelaccordingtothemanufacturer’sinstructions.Alternatively,verticalnon-denaturingpolyacrylamidegelelectrophoresiscanbeperformed,forexampleasdescribedin.

RemovethegelfromthePhastSystemandimagewithasuitableimagingtechnique(autoradiographywithaPhosphorImagerinthecaseofradioactiveband-shifts;fluorescenceimaginginthecaseoffluorescentband-shifts;.

Band-shiftassayforthedeterminationofKd

Determinationoftheconcentrationofradiolabeledantibody

Inparalleltubes,incubatea‰1µMradiolabeledantibodysolutioninPBSwithincreasingconcentrationsofantigen(e.g.,rangingbetween0.1µMand10µM).Atthesehighconcentrations(>Kd),theantibodyshouldrapidlyandquantitativelybindtotheavailableantigen.

Afterfewminutesofincubation,runthereactionmixturesonanon-denaturinggelasdescribedintheprevioussection.

Imagethegelbyautoradiography(preferablyonaPhosphorImager).Theconcentrationofantigenatwhicha"bandshift"isobserved(Figure1)isequaltotheconcentrationofradiolabeledantibodyusedintheassay(N.B.:itisNOTequaltotheKd,sinceweareworkinginaconcentrationrange>Kd).

Determinationofthedissociationconstant

Intheseexperiments,itisconvenienttoworkatantibodyconcentrationslowerthanthedissociationconstant.Thisrangeofconcentrationsistypicallynotknownapriori,andisdeterminedbyperformingband-shiftexperimentsatlowantibodyconcentrations(e.g.,0.1—1nM).Theintensityofthebandsisintegratedwithappropriatemethodsandfittedtotheequation:Kd=[A][B]/[AB](thisprocedurereliesontheassumptionthatbandintensityisproportionaltotheconcentrationsatthemomentthesampleisloadedonthegel).ThisfittingmayyieldanacceptableKdvalue,ormayindicateinwhichconcentrationrangeoneshouldworktoobtainmoreaccurateresults.Anotherimportantissuetoconsiderishowlongshouldantibodyandantigenbeallowedtoreactbeforeanalysingthereactionmixtureonanativegel.Inpseudo-firstorderconditions(e.g.,whentheantibodyconcentrationiskeptconstantand<<Kd)thisreactiontimeisafunctionofthekineticassociationconstantk_onandoftheantigenconcentration.Theconcentrationsofantibodyfreeandincomplexwillexponentiallytendtoalimitvalue,andoneshouldwaitatleastt>1/(k_onx[Antigen]_total)beforeconsideringthereactionclosetocompletion.Wheneverthek_onoftheantibodyisnotknown,itisimportanttomeasureaffinityconstantsbyband-shiftwithtwodifferentincubationtimes.Ifthereactionhasreachedequilibrium,theaffinityvaluesinthetwoexperimentsshouldnotdiffersignificantly.TheprotocollistedbelowisforalabeledantibodywithKd=3nMandk_on=10⁶s^-1M^-1.

Inparalleltubes,incubatea0.3nMradiolabeledantibodysolutionwithincreasingconcentrationsofantigen(e.g.,rangingbetween0.3nMand30nM)inPBS.

After30minutesincubationatroomtemperature,runthereactionmixturesonanon-denaturinggelasdescribedintheprevioussection.

Imagethegelbyautoradiography(preferablyonaPhosphorImager).Theconcentrationofantigenatwhicha"bandshift"isobserved(Figure1)isequaltothedissociationconstantKd.Alternatively,theKdvaluecanbeobtainedbyfittingtheintensityofthebandstotheequation:Kd=[A][B]/[AB].

Tobesurethattheequilibriumisreachedwhenthesamplesareappliedontothegel,repeattheprocedure,with2hoursincubationtimeinstep2.

Band-shiftassayforthedeterminationofk_offAprerequisiteofthistechniqueisthattheantibody-antigencomplexcanbedetectedinanon-denaturinggel(ifpossibleasasingleband).Iftheantigenisfluorescentlylabeled,itisessentialtobeabletoresolvethebandofthecomplexfromthebandofthefreeantigen.Itisalsoworthmentioningthatonlynegativelychargedproteins(orproteincomplexes)migratetowardstheanodeinastandardnon-denaturingpolyacrylamidegelwithTris/Glycinebuffer,pH8.3(seeabove).Ifonewishestostudypositivelychargedproteins(orproteincomplexes),oneneedstoworkwithreversepolarityelectrodesandpossiblywithbuffersystemsdifferentfromTris/Glycine(seemanufacturer’sinstructionsofthePhastSystem,AmershamPharmaciaBiotech).Thefollowingprotocoliswrittenforthenegativelychargedcomplexofarecombinantantibodywithafluorescentlylabeledantigen.Althoughthereactionconditionsareheregiveninthemicromolarrange,muchlowerconcentrationscanalsobeusedifahigh-sensitivityluminescenceanalyserisavailable(seeforexample.

1. Inseparatereactiontubes,incubatea1µMsolutionoffluorescentlylabeledantigenwitha1µMsolutionofantibodyinPBSforfewminutesatroomtemperature.
2. Atdifferenttimes(e.g.,attime0h,24h,48h,60h,66h,69h,71h,71h30min,71h40min;thechoiceoftimeintervalsdependsonthek_offvalue)addunlabeledantigentooneofthetubes,reachingafinalantigenconcentrationof20µM.Bythisprocedure,inthedifferenttubestheantibody-(labeledantigen)complexisallowedtocompetewithamolarexcessofunlabeledantigenfordifferentamountsoftime.
3. Attheendofthecompetitionreactions(e.g.,at71h41min),runthereactionmixturesonanon-denaturinggelasdescribedinaprevioussection.
4. Imagethegelwithasuitablefluorescenceimager,makingsurethateventhestrongestbandsfallinthelineardetectionrangeoftheimager.
5. Integratethefluorescenceintensityofthebands.
6. Plotbandvolumeversustime(Figure3).Anexponentialfitofthebandvolumedecayingwithtimewillyieldthecharacteristicconstantk_off.

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