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Antibodies-Online/GFP-Trap® M Kit/ABIN509406/20 tests

Antigen
GreenFluorescentProtein(GFP)
  • greenfluorescentprotein
  • gfp
Reactivity
Aequoreavictoria
11Aequoreavictoria
Host
Camelidae
AntibodyType
RecombinantAntibody
Conjugate
MagneticParticles
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(magneticparticles)forbiochemicalanalysisofGFPfusionproteinsandtheirinteractingpartners.
BrandGFP-Trap®
SampleTypeCellExtracts
SpecificityBindingcapacity:10µLGFP-Trap®_Mslurrybinds0.25-0.5µgofGFP
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®coupledtomagneticparticles
Thekitisincludinglysis,washandelutionbuffers.
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

Beadsize0.5-1µm

AssayTime1.5h
Protocol
  • RobustandversatiletoolforbiochemicalanalysesofGFP-fusionproteins
  • Shortincubationtimes(5-30min)
  • Quantitativeisolationoffusionproteinsandtransientlyboundfactorsfromcellextractsororganelles
  • Lowunspecificbinding
  • Nocontaminatingheavyandlightchainsofconventionalantibodies
  • ApplicableinChromatinImmunoprecipitation(ChIP)
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°C.Forimmunoblotanalysisdilute50µLcelllysatewith50µL2xSDS-samplebuffer(àrefertoasinput).
  • 5.EquilibrateGFP-Trap®_Mbeadsindilutionbuffer.Resuspendmagneticbeadsbyvortexingandtransfer20-30µLbeadslurryin500µLicecolddilutionbuffer.Magneticallyseparatebeadsuntilsupernatantisclear.Discardsupernatantandwashbeads2moretimeswith500µLicecolddilutionbuffer.
  • 6.AddcelllysatetoequilibratedGFP-Trap®_MbeadsandincubatetheGFPTrap®_Mbeadswiththecelllysateunderconstantmixingfor10min–2hatroomtemperatureor4°C.
    note:duringincubationofproteinsamplewiththeGFP-Trap®_Mthefinalconcentrationofdetergentsshouldnotexceed0.2%toavoidunspecificbindingtothematrix.
  • 7.Magneticallyseparatebeadsuntilsupernatantisclear.Forwesternblotanalysisdilute50µLsupernatantwith50µL2xSDS-samplebuffer(àrefertoasnonbound).Discardremainingsupernatant.
  • 8.Washbeadsthreetimeswith500µLicecoldwashbuffer.Afterthelastwashstep,transferbeadstonewtube.
    optional:increasesaltconcentrationinthesecondwashingstepupto500mM
  • 9.ResuspendGFP-Trap®_Mbeadsin100µL2xSDS-Samplebufferorgotostep11.
  • 10.Boilresuspendedbeadsfor10minutesat95°Ctodissociatetheimmunocomplexesfromthebeads.ThebeadscanbemagneticallyseparatedandSDS-PAGEisperformedwiththesupernatant(àrefertoasbound).
  • 11.optional:eluteboundproteinsbyadding50µL0.2MglycinepH2.5(incubationtime:30secunderconstantmixing)followedbycentrifugation.Transferthesupernatanttoafreshcupandadd5µL1MTrisbase(pH10.4)forneutralization.Toincreaseelutionefficiencythisstepcanberepeated.

RestrictionsForResearchUseonly
Concentration500µLresin
Buffer1xPBS,0.01%Sodiumazide
PreservativeSodiumazide
PrecautionofUseThisproductcontainssodiumazide:aPOISONOUSANDHAZARDOUSSUBSTANCEwhichshouldbehandledbytrainedstaffonly.
HandlingAdviceDonotfreeze.
Storage4°C
ExpiryDate12months
SupplierImages
image for GFP-Trap® M Kit (ABIN509406)Left(IP):PulldownofGFPwithGFP-Trap®_AandGFP-Trap®_Mfrom293Tcellextracts....
Productcitedin: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."in:PLoSpathogens,Vol.11,Issue5,pp.e1004909,2015(PubMed).

Vardabasso,Gaspar-Maia,Hasson,Pünzeler,Valle-Garcia,Straub,Keilhauer,Strub,Dong,Panda,Chung,Yao,Singh,Segura,Fontanals-Cirera,Verma,Mann,Hernando,Hake,Bernstein:"HistoneVariantH2A.Z.2MediatesProliferationandDrugSensitivityofMalignantMelanoma."in:Molecularcell,Vol.59,Issue1,pp.75-88,2015(PubMed).

Mochida,Oikawa,Kimura,Kirisako,Hirano,Ohsumi,Nakatogawa:"Receptor-mediatedselectiveautophagydegradestheendoplasmicreticulumandthenucleus."in:Nature,Vol.522,Issue7556,pp.359-62,2015(PubMed).

Voit,Seiler,Grummt:"CooperativeActionofCdk1/cyclinBandSIRT1IsRequiredforMitoticRepressionofrRNASynthesis."in:PLoSgenetics,Vol.11,Issue5,pp.e1005246,2015(PubMed).

VanItallie,Tietgens,Krystofiak,Kachar,Anderson:"AcomplexofZO-1andtheBAR-domainproteinTOCA-1regulatesactinassemblyatthetightjunction."in:Molecularbiologyofthecell,Vol.26,Issue15,pp.2769-87,2015(PubMed).

Ojeda,Robles-Valero,Barreira,Bustelo:"Thedisease-linkedGlu-26-LysmutantversionofCoronin1Aexhibitspleiotropicandpathway-specificsignalingdefects."in:Molecularbiologyofthecell,Vol.26,Issue16,pp.2895-912,2015(PubMed).

Catinot,Huang,Huang,Tseng,Chen,Gu,Lo,Wang,Chen,Zimmerli:"ETHYLENERESPONSEFACTOR96positivelyregulatesArabidopsisresistancetonecrotrophicpathogensbydirectbindingtoGCCelementsofjasmonate-andethylene-responsivedefencegenes."in:Plant,cell
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