| AssayProcedure | Asanexamleprotocolwewouldmentionthefollowing:
II.QuickStartHintsandTips
InfraredfluorescencedetectionwithOdysseyFamilyImagingSystemsprovidesaquantitativetwo-colordetectionmethodforWesternblots.Following,youwillfindsomebasicHintsandTipstohelpyougetstarted.
1.StoretheIRDyesecondaryantibodyvialindarknessat4 °C.Minimizeexposuretolightandtakecarenottointroducecontaminationintothevial.Diluteimmediatelypriortouse.Ifparticulatesareseenintheantibodysolution,centrifugebeforeuse.
Page2-Near-Infrared(NIR)WesternBlotDetection
2.Thebesttransferconditions,membrane,andblockingagentforexperimentswillvary,de-pendingontheantigenandantibody.
3.DonotwriteonblotwithapenorSharpie®Marker.Inkfrommostpensandmarkerswillfluoresceinthe700nmchannelofallOdyssey®FamilyImagingSystems.Theinkmaywashoffandre-depositelsewhereonthemembrane,causingincreasedbackground.Useapenciltomarkmembranes.InkfromtheOdysseyPendoesnotfluoresceandcanbeusedtomarknitrocellulosemembranes.IftheOdysseyPenisusedforPVDFmembranes,theinkwilldissolveandwashoffwhentheblotiswettedinmethanol.
4.Letthemembranedryaftertransferfor1hourorovernight,tomaximizeproteinretentiononthemembrane.
5.Handleblotwithcleanforcepsonly.
6.Beforeusingforceps,incubationtrays,andtheOdysseyscanningsurfaceorsampletray(ifapplicable),cleanwith100 %methanoltoremoveanyresidualdyesignalfromprevioususe.Rinsewithasmallvolumeofdistilledwater,followedbyisopropanol.Drywithalint-freewipe.
7.WhenprocessingWesternblots,donotusedishes/boxesthathaveeverbeenusedforCoomassiestaining.TheOdysseyimagersareverysensitivetoCoomassie(whichisastrongly-fluorescentdye),anduseofdisheswithsmalltracesofCoomassiewilladdatremendousamountofbackgroundinthe700nmchannel.
MaintainthesamebuffersystemthroughouttheWesternblotprocess.Forexample,ifyoublockyourblotinOdysseyBlockingBuffer(PBS),usePBS-basedbuffersthroughouttheprotocol.
8.Donotincludedetergentsduringtheblockingstep.
9.For1-colorblots,useIRDye®800CWsecondaryantibodyfordetectionoftheproteinforbestsensitivity.
10.For2-colorblots:
Makesureprimaryantibodiesarefromdifferentspecies(forexample,RabbitandMouse)
UsetheIRDye800CWsecondaryantibodytodetecttheproteintargetwithlowestabundance,andIRDye680RDsecondaryantibodytodetectthemoreabundantprotein.
11.IfyouareusingPVDF,add0.01 %SDStothedilutedsecondaryantibody.DonotaddSDSifusingnitrocellulosemembrane.
12.Incubatewithsecondaryantibodiesinthedarkforonehourwithgentleshaking.Theincu-bationboxcanbecoveredwithaluminumfoil.
Near-Infrared(NIR)WesternBlotDetection
III.WesternBlotDetectionMethods
ThisprotocolisdesignedtohelpyouachievesuccesswithNIRWesternblotdetectionmethods.Readtheentireprotocolcarefullybeforebeginningyouroptimizationexperiments.
MembraneGuidelines
Alow-backgroundmembraneisessentialforNIRWesternblotsuccess.Backgroundcanresultfrommembraneautofluorescenceorfromnon-specificbindingofantibodies.Polyvinylidenefluo-ride(PVDF)andnitrocellulosemembranesaretypicallyusedforWesternblottingapplications.Therearemanybrandsandvendorsforbothtypesofmembrane.
BeforeusingyourblottingmembraneforthefullOdyssey®Westernblotprotocol,cutasmallsampleofmembranefortesting.Imagethissample(bothwetanddry)toevaluatethelevelofmembraneautofluorescence.Ifpossible,includeasampleofmembranethatisknowntoworkwellwiththeOdysseysystem,soyoucancomparebackgroundlevels.
TolearnmoreaboutoptimizingyourWesternblots,seeGoodWesternsGoneBad:TipstoMakeYourNIRWesternBlotGreat(www.licor.com/GWGBIR)
WesternBlotDetectionProtocol
Aftermembranetransfer,performthefollowingsteps:
1.ForPVDFmembrane:
a.Pre-wet1 minutein100 %methanol.
b.Rinsewithultrapurewater.
c.Wetin1XPBSorTBSfor2 minutes(usingtheappropriatebuffersystem).
Fornitrocellulosemembrane:
a.Wetin1XPBSorTBSfor2 minutes,oruntilfullyhydrated(usingtheappropriatebuffersystem).
2.PlacemembraneinincubationboxandblockthemembraneinOdysseyBlockingBuffer(PBSorTBS)for1hourwithgentleshaking.Besuretousesufficientblockingbuffertocoverthemembrane(aminimumof0.4 mL/cm2issuggested).
3.Prepareprimaryantibody:
a.Primaryantibodydiluent:OdysseyBlockingBuffer(PBSorTBS)+0.2 %Tween®20(finalconcentration).
b.DiluteprimaryantibodyinOdysseyBlockingBuffer+0.2 %Tween20,usingtheven-dorsrecommendeddilutionforWesternblotapplications.Dilutionstypicallyrangefrom1:200-1:5,000,dependingontheprimaryantibody.
c.Useenoughantibodysolutiontocompletelycoverthemembrane.
4.Incubateblotindilutedprimaryantibodyfor1to4hoursatroomtemperaturewithgentleshaking,orovernightat4 °C.
Optimalincubationtimesvaryfordifferentprimaryantibodies.
Iftheprocedurecannotbecompletedinfull,thisisagoodplacetostopuntilthefollowingday.Incubatetheblotinprimaryantibodyovernightat4 °Cwithgentleshaking,andre-sumetheprotocolthenextday.
5.Washmembranes:
•Pouroffprimaryantibodysolution.
•Rinsemembranewithappropriatebuffer-1XTBS-T(0.1 %Tween®20)or1XPBS-T(0.1 %Tween20).
•Coverblotwith1XTBS-T(0.1 %Tween20)or1XPBS-T(0.1 %Tween20).
•Shakevigorouslyonplatformshakeratroomtemperaturefor5 minutes.
•Pouroffwashsolution.
•Repeat3additionaltimes.
6.DiluteIRDye®secondaryantibodyintheappropriatediluentlistedbelow:
a.Secondaryantibodydiluent:PVDFmembrane
•Toblockingbuffer,addTween20toafinalconcentrationof0.2 %andSDStoafinalconcentrationof0.01 %.
b.Secondaryantibodydiluent:nitrocellulosemembrane
•Toblockingbuffer,addTween20toafinalconcentrationof0.2 %.DonotaddSDS.
NOTE:ASuggesteddilutionrangeforsecondaryantibodiesistypically1:5,000to1:25,000.Recommendeddilutionscanbefoundonthesecondaryantibodypackinsert.Use1:20,000asasuggestedstartingpointifusingLI-CORsecondaryantibodies.
7.Protectmembranefromlightduringincubation.Incubateblotindilutedsecondaryantibodyforonehouratroomtemperaturewithgentleshaking.
8.Protectmembranefromlightduringwashes.
Washmembranes:
•Pouroffsecondaryantibodysolution.
•Rinsemembranewiththeappropriatebuffer-1XTBS-T(0.1 %Tween20)or1XPBS-T(0.1 %Tween20).
•Coverblotwith1XTBS-T(0.1 %Tween20)or1XPBS-T(0.1 %Tween20).
•Shakevigorouslyonplatformshakeratroomtemperaturefor5 minutes.
•Pouroffwashsolution.
•Repeat3additionaltimes.
9.Rinsemembranewith1XTBS/PBS(asappropriate)toremoveresidualTween20.
10.TheWesternblotisnowreadytoimage.
•Themembranecanbestoredin1XTBSor1XPBSforupto48hoursinthedarkat4 °C.
Iftheblotispreparedmorethan48hoursinadvance,air-drytheblotandstoreinthedarkatroomtemperatureuntilreadytoimage.
11.ImagewithanOdyssey®FamilyImagingSystem.
•Ifsignalonmembraneistoostrongortooweak,adjusttheimagingparameterstoopti-mizetheimage.
-OdysseyClassic:Re-imagethemembraneatalowerorhigherscanintensitysetting,respectively.
-OdysseyFc:Adjustimageacquisitiontime.
-OdysseyCLx:UsetheAutoScanfunctiontoimprovethedynamicrangeoftheimage.
IV.GuidelinesforTwo-ColorDetection
TwodifferentantigenscanbedetectedsimultaneouslyonthesameblotusingIRDye®secondaryantibodies.Whenperformingatwo-colorblot,usethestandardWesternblotprotocolwiththefollowingmodifications:
•Combinethetwoprimaryantibodiesinthedilutedantibodysolution(Step3,SectionIII).Incubatesimultaneouslywithmembrane(Step4,SectionIII).Theprimaryantibodiesmustbefromtwodifferenthostspecies.
•CombinethetwoIRDye®secondaryantibodiesinthedilutedantibodysolution(Step6,SectionIII).Incubatesimultaneouslywithmembrane(Step7,SectionIII).
Two-colordetectionrequirescarefulselectionofprimaryandsecondaryantibodies.Thefollowingguidelineswillhelpyousuccessfullydesigntwo-colorexperiments:
•Thetwoprimaryantibodiesmustbederivedfromdifferenthostspeciessothattheycanbediscriminatedbysecondaryantibodiesofdifferentspecificities(forexample,primaryanti-bodiesfromrabbitandmousewillbediscriminatedbyanti-rabbitIgGandanti-mouseIgGsecondaryantibodies,respectively).
•IfthetwoprimaryantibodiesaremousemonoclonalsfromdifferentIgGsubclasses(IgG1,IgG2a,orIgG2b),IRDyeSubclass-Specificsecondaryantibodiescanbeusedformultiplexdetection.Thesamesubclassescannotbecombinedinatwo-colorWesternblot(forexam-ple,twoIgG1primaryantibodies).Fordetails,refertoWesternBlotandIn-CellWestern™AssayDetectionUsingIRDyeSubclass-SpecificAntibodies
•Anti-GoatsecondaryantibodiescannotbemultiplexedwithGoat-derivedsecondaryanti-bodies(Example:Donkeyanti-GoatandGoatanti-Rabbit).Thesecondaryantibodieswillcross-react.
•Onesecondaryantibodymustbelabeledwitha700nmchanneldyeandtheotherwith800nmchanneldye.
•Ingeneral,itisrecommendedthattheIRDye®800CWsecondaryantibody(800nmchannel)beusedtodetectthelower-abundanceproteintargetandIRDye680RDsec-ondaryantibody(700nm)todetectthemoreabundantprotein.
•Alwaysusehighlycross-adsorbedsecondaryantibodiesfortwo-colordetection.Failuretousecross-adsorbedantibodiesmayresultinincreasedcross-reactivityofthesecond-aryantibodies.
•Forbestresults,avoidusingprimaryantibodiesfrommouseandrattogetherinatwo-colorexperiment.Thetwospeciesaresocloselyrelatedthatitisnotpossibletocom-pletelyadsorbawayallcross-reactivity.Ifthereisnootheroption,itiscrucialtorunsin-gle-colorblotsfirstwitheachindividualantibodytobecertainofexpectedbandsizes.
V.AdaptingWesternBlotProtocolsforOdyssey®ImagingSystemsWhenadaptingWesternblottingprotocolsforOdysseydetectionorusinganewprimaryanti-body,itisimportanttodeterminetheoptimalantibodyconcentrations.Optimizationwillhelpachievemaximumsensitivityandconsistency,whileminimizingbackground.Threeparametersshouldbeoptimized:primaryantibodyconcentration,IRDyesecondaryantibodyconcentration,anddetergentconcentrationinthedilutedantibodies.
PrimaryAntibodyConcentration
Primaryantibodiesvarywidelyinquality,affinity,andconcentration.Thecorrectworkingrangeforantibodydilutiondependsonthecharacteristicsoftheprimaryantibodyandtheamountoftargetantigentodetect.StartwiththevendorsdilutionrecommendationforWesternblottingorwiththedilutionnormallyusedforchemiluminescentdetection.
SecondaryAntibodyConcentration
OptimaldilutionsofIRDyesecondaryantibodiesshouldalsobedetermined.Refertotheappro-priatepackinsertforrecommendationsathttp://www.licor.com/packinsert.Theamountofsec-ondaryantibodyrequiredvariesdependingonhowmuchantigenisbeingdetected.Abundantproteinswithstrongsignalsmayrequirelesssecondaryantibody.Usingtoomuchsecondaryantibodymayincreasemembranebackgroundand/ornon-specificbanding.
DetergentConcentration
Additionofdetergentstodilutedantibodiescansignificantlyreducebackgroundontheblot.Optimaldetergentconcentrationwillvary,dependingontheantibodies,membranetype,andblockerused.Keepinmindthatsomeprimaryantibodiesdonotbindastightlyasothersandmaybewashedawaybytoomuchdetergent.Neverexposethemembranetodetergentuntilblockingiscomplete,asthismaycausehighmembranebackground.
Tween®20:
•Blockingbuffer-donotaddTween20duringblocking.
•DilutedprimaryandsecondaryantibodiesshouldcontainTween20.
Useafinalconcentrationof0.1-0.2 %Tween20forPVDFornitrocellulosemembranes.
•Washsolutionsshouldcontain0.1 %Tween20.
SDS:
WhenusingPVDFmembrane,additionofSDSwilldramaticallyreduceoverallmem-branebackgroundinthe700nmchannel.Itiscriticaltouseonlyaverysmallamount,becauseSDSisanionicdetergentandcandisruptantibody-antigeninteractionsiftoomuchispresentatanytimeduringtheprotocol.SDSshouldnotbeusedwithnitrocellulosemembranes.
•Blockingbuffer-donotaddSDStotheblockingreagentduringblocking.
•DilutedprimaryantibodiesshouldnotcontainSDS.
•Dilutedsecondaryantibodiesshouldcontainafinalconcentrationof0.01-0.02 %SDSand0.1-0.2 %Tween®20,whenPVDFmembraneandIRDye®680LTsecondaryantibodiesareused.
•SDSisoptionalwhenusingIRDye680RDantibodieswithPVDFmembrane,butisessentialwhenusingIRDye680LTantibodieswithPVDF.
•WashsolutionsshouldnotcontainSDS.
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