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AntibodiesOnline/GFPTrap® A/ABIN509400/400 tests
Antigen
GreenFluorescentProtein(GFP)
  • greenfluorescentprotein
  • gfp
Reactivity
Aequoreavictoria
11Aequoreavictoria
Host
Camelidae
AntibodyType
RecombinantAntibody
Conjugate
AgaroseBeads
Application
AffinityMeasurement(AM),ChromatinImmunoprecipitation(ChIP),EnzymeActivityAssay(EAA),Immunoprecipitation(IP),MassSpectrometry(MS),ProteinComplexImmunoprecipitation(Co-IP),Pull-DownAssay(Pull-Down),Purification(Purif)
Options
Bulkdiscount
Supplier
SupplierProductNo.
PurposeGFP-Trap®isahighqualityGFP-bindingproteincoupledtoamonovalentmatrix(agarosebeads)forbiochemicalanalysisofGFPfusionproteinsandtheirinteractingpartners.
BrandGFP-Trap®
SampleTypeCellExtracts
SpecificityBindingcapacity:10µLGFP-Trap®_Aslurrybinds2.5-3µgofGFP
Cross-Reactivity(Details)GFP-Trap®specificallybindstoeGFP,wtGFP,GFPS65T,TagGFP,eYFP,YFP,Venus,Citrin,CFP.NobindingtoproteinsderivedfromDsRed,allRFPsandTurboGFPcanbedetected.
CharacteristicsAntibodies-extremelypowerfultoolsinbiomedicalresearch-arelargecomplexmolecules(~150kDa)consistingoftwoheavyandtwolightchains.Duetotheircomplexstructure,theuseofantibodiesisoftenlimitedandhinderedbybatch-to-batchvariations.

Camelidae(camels,dromedaries,llamasandalpacas)possessfunctionalantibodiesdevoidoflightchains,so-calledheavychainantibodies(hcAbs).hcAbsrecognizeandbindtheirantigensviaasinglevariabledomain(VHH).TheseVHHdomainsarethesmallestintactantigenbindingfragments(~13kDa).

Nano-Trapsarebasedonsingledomainantibodyfragments(VHHs)derivedfromalpaca.
ComponentsGFP-Trap®coupledtoagarosebeads
MaterialnotincludedLysisbuffer(CoIP),10xRIPAbuffer,Dilutionbuffer,Washbuffer,Elutionbuffer
AlternativeNameGFP
BackgroundThegreenfluorescentprotein(GFP)andvariantsthereofarewidelyusedtostudythesubcellularlocalizationanddynamicsofproteins.GFPfusionproteinscanbeexpressedindifferentcelltypesatdifferentexpressionlevelsbytransientorstabletransfection.Transientexpressionmayprovidequickinformativeresults,however,inmanycasesitisnecessarytogeneratestablecelllinesthatexpresstheGFPfusionproteinofinterestatalevelsimilartotheoneoftheendogenousprotein.QuantificationofGFPfusionproteinsincellscanbetrickysinceexistingmethods,likefluorescencemicroscopyorWesternBlotting,areoftenshowsinsufficientsignaltonoiseratiosorhighsignalvariABIlities.
ResearchAreaTags/Labels
ApplicationNotesGreenfluorescentproteins(GFP)andvariantsthereofarewidelyusedtostudyproteinlocalizationanddynamics.ForbiochemicalanalysesincludingmassspectroscopyandenzymeactivitymeasurementstheseGFP-fusionproteinsandtheirinteractingfactorscanbeisolatedfastandefficiently(onestep)viaImmunoprecipitationusingtheGFP-Trap®.TheGFP-Trap®_AenablespurificationofanyproteinofinterestfusedtoGFP.
Comment

Beadsize~90µm

AssayTime1.5h
Protocol
  • RobustandversatiletoolforbiochemicalanalysesofGFP-fusionproteins
  • Shortincubationtimes(5-30min)
  • Quantitativeisolationoffusionproteinsandtransientlyboundfactorsfromcellextractsororganelles
  • Lowunspecificbinding
  • Nocontaminatingheavyandlightchainsofconventionalantibodies
  • ApplicableinChromatinImmunoprecipitation(ChIP)
ReagentPreparation

Suggestedbuffercomposition

  • Lysisbuffer(CoIP):10mMTris/ClpH7.5,150mMNaCl,0.5mMEDTA,0.5%NP-40
  • 10xRIPAbuffer:10mMTris/ClpH7.5,150mMNaCl,5mMEDTA,0.1%SDS,1%TritonX-100,1%Deoxycholate
  • Dilutionbuffer:10mMTris/ClpH7.5,150mMNaCl,0.5mMEDTA
  • Washbuffer:10mMTris/ClpH7.5,150mMNaCl,0.5mMEDTA
  • Elutionbuffer:200mMglycinepH2.5

AssayProcedure

Beforeyoustart:Add1mlPBStoyourcellsandscrapethemoffthepetridish.Transfertoprecooledtube,spin3minat500xganddiscardsupernatant.WashcellpellettwicewithicecoldPBS,brieflyresUSPendingthecells.

  • 1.Foroneimmunoprecipitationreactionresuspendcellpellet(~10^7mammaliancells)in200µLlysisbufferbypipetting(orusingasyringe).
    optional:add1mMPMSFandProteaseinhibitorcocktail(notincluded)tolysisbuffer
    optionalfornuclear/chromatinproteins:add1mg/mlDNaseand2.5mMMgCl2(notincluded)tolysisbuffer
  • 2.Placethetubeonicefor30minwithextensivelypipettingevery10min.
  • 3.Spincelllysateat20.000xgfor5-10minutesat4°C.
  • 4.Transfersupernatanttoapre-cooledtube.Adjustvolumewithdilutionbufferto500µL–1000µL.Discardpellet.
    optional:add1mMPMSFandProteaseinhibitorcocktail(notincluded)todilutionbuffer
    note:thecelllysatecanbefrozenatthispointforlong-termstorageat-80°CForimmunoblotanalysisdilute50µLcelllysatewith50µL2xSDS-samplebuffer(àrefertoasinput).
  • 5.EquilibrateGFP-Trap®_Abeadsindilutionbuffer.Resuspend20-30µLbeadslurryin500µLicecolddilutionbufferandspindownat2.500xgfor2minutesat4°C.Discardsupernatantandwashbeads2moretimeswith500µLicecolddilutionbuffer.
  • 6.AddcelllysatetoequilibratedGFP-Trap®_AbeadsandincubatetheGFP-Trap®_Abeadswiththecelllysateunderconstantmixingfor10min–2hatroomtemperatureor4°C.
    note:duringincubationofproteinsamplewiththeGFP-Trap®_Athefinalconcentrationofdetergentsshouldnotexceed0.2%toavoidunspecificbindingtothematrix
  • 7.Spintubeat2.500xgfor2minutesat4°C.Forwesternblotanalysisdilute50µLsupernatantwith50µL2xSDS-samplebuffer(àrefertoasnon-bound).Discardremainingsupernatant.
  • 8.Washbeadsthreetimeswith500µLicecoldwashbuffer.Afterthelastwashstep,transferbeadstonewtube.
    optional:increasesaltconcentrationinthesecondwashingstepupto500mM
  • 9.ResuspendGFP-Trap®_Abeadsin100µL2xSDS-Samplebufferorgotostep11.
  • 10.Boilresuspendedbeadsfor10minutesat95°Ctodissociatetheimmunocomplexesfromthebeads.Thebeadscanbecollectedbycentrifugationat2.500xgfor2minutesat4°CandSDS-PAGEisperformedwiththesupernatant(àrefertoasbound).
  • 11.optional:eluteboundproteinsbyadding50µL0.2MglycinepH2.5(incubationtime:30secunderconstantmixing)followedbycentrifugation.Transferthesupernatanttoafreshcupandadd5µL1MTrisbase(pH10.4)forneutralization.Toincreaseelutionefficiencythisstepcanberepeated.

RestrictionsForResearchUseonly
Concentration2.5mLresin
Buffer20%EtOH
HandlingAdviceDonotfreeze.
Storage4°C
ExpiryDate12months
SupplierImages
image for GFP-Trap® A (ABIN509397)Left(IP):PulldownofGFPwithGFP-Trap®_AandGFP-Trap®_Mfrom293Tcellextracts....
Western Blotting (WB) image for GFP-Trap® A (ABIN509397)ComparisonofGFP-Trapwithconventionalmono-andpolyclonalantibodies:Immunopreci...
Productcitedin:Braiterman,Gupta,Chaerkady,Cole,Hubbard:"CommunicationbetweentheN-andC-terminiisrequiredforCu-stimulatedSer/ThrphosphorylationofCu-(I)ATPase(ATP7B)."in:TheJournalofBIOLOGicalchemistry,2015(PubMed).

Yan,Chu,Qin,Wang,Liu,Jin,Zhang,Gomez,Hergovich,Chen,He,Gao,Yao:"RegulationofNDR1activitybyPLK1ensuresproperspindleorientationinmitosis."in:Scientificreports,Vol.5,pp.10449,2015(PubMed).

Xiao,MacNair,McGoldrick,McKeever,McLean,Zhang,Keith,Zinman,Rogaeva,Robertson:"IsoformSpecificAntibodiesRevealDistinctSubcellularLocalizationsofC9orf72inAmyotrophicLateralSclerosis."in:Annalsofneurology,2015(PubMed).

Satpathy,Wagner,Beli,Gupta,Kristiansen,Malinova,Francavilla,Tolar,Bishop,Hostager,Choudhary:"Systems-wideanalysisofBCRsignalosomesanddownstreamphosphorylationandubiquitylation."in:Molecularsystemsbiology,Vol.11,Issue6,pp.810,2015(PubMed).

Hyodo,Taniguchi,Manabe,Kaido,Mise,Sugawara,Taniguchi,Okuno:"PhosphatidicacidproducedbyphospholipaseDpromotesRNAreplicationofaplantRNAvirus.

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