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AntibodiesOnline/JNK ELISA Kit (MitogenActivated Protein Kinase 8)/ABIN4881289/5 x 96 tests188bio精品生物—专注于实验室精品爆款的电商平台 - 蚂蚁淘旗下精选188款生物医学科研用品
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AntibodiesOnline/JNK ELISA Kit (MitogenActivated Protein Kinase 8)/ABIN4881289/5 x 96 tests
Antigen
Mitogen-ActivatedProteinKinase8(MAPK8)
  • BSK
  • BSK/DJNK
  • Bsk
  • CG5680
  • D-JNK
  • D-junk
  • DBSK/JNK
  • DJNK
  • DJNK/bsk
  • DmelCG5680
  • JNK
  • JNK/SAPK
  • Jnk
  • Junk
  • SAPKa
  • c-Jun
  • dJNK
  • jnk
  • JNK-46
  • JNK1
  • JNK1A2
  • JNK21B1/2
  • PRKM8
  • SAPK1
  • SAPK1c
  • AI849689
  • Prkm8
  • jnk1
  • sapk1
  • T10F20.15
  • mapk8
  • zgc:112379
  • basket
  • mitogen-activatedproteinkinase8
  • mitogen-activatedproteinkinase8b
  • ProteinJNK-1
  • bsk
  • MAPK8
  • Mapk8
  • mapk8
  • ATMPK8
  • mapk8b
  • jnk-1
Alternatives
HumanJNKELISAKit
Kitswithalternativereactivityto:
18Human
10Mouse(Murine)
10Rat(Rattus)
2Mammalian
MethodType
CellELISA
Application
ELISA
Options
Bulkdiscount
Supplier
SupplierProductNo.
PurposeCell-BasedHuman/Mouse/RatJNK(Thr183/Tyr185)PhosphorylationELISAKit.Suitableforadherentwholecelllines.
BrandCellBIND®
SampleTypeCellCultureCells
AnalyticalMethodSemi-Quantitative
DetectionMethodColorimetric
SpecificityTheantibodiesprovidedinthiskitrecognizeshuman,mouseandratJNKphosphorylatedatsitesThr183andTyr185aswellastotalJNKforcomparison.
Characteristics
  • Siteandsignalpathway-specific
  • Invitrodetectionofadherentcellculture
  • Nosamplelysisneeded
  • CompatiblewithastandardELISAplatereader
  • FasterresultsthanwithELISA
  • Adaptableforhigh-throughputscreeninganddrugdiscovery
Components
  • uncoated96-wellMicroplate
  • WashBufferA
  • WashBufferB
  • FixingSolution
  • QuenchingBuffer
    • BlockingBuffer
  • Anti-phosphoantibody
  • Anti-panantibody
  • HRP-ConjugatedSecondaryAntibody
  • TMBOne-StepSubstrate
  • StopSolution
    • Materialnotincluded
    • Distilledordeionizedwater
    • 100 mLand1litergraduatedcylinders
    • Tubestopreparesampledilutions
    • ProteaseandPhosphataseinhibitors
    • Precisionpipettestodeliver2 μLto1 mLvolumes
    • Adjustable1-25 mLpipettesforreagentpreparation
    • Benchtoprockerorshaker
    • Microplatereadercapableofmeasuringabsorbanceat450nm
    AlternativeNameJNK(MAPK8ELISAKitAbstract)
    GeneID5599
    UniProtP45983
    PathwaysMAPKSignaling,WNTSignaling,TLRSignaling,Fc-epsilonReceptorSignalingPathway,NeurotrophinSignalingPathway,ActivationofInnateimmuneResponse,HepatitisC,Toll-LikeReceptorsCascades,SignalingofHepatocyteGrowthFactorReceptor
    SampleVolume100μL
    PlateUncoated
    Protocol
    1. Seed10,000-30,000cellsintoeachwellandincubateovernight.
    2. Applyvarioustreatment,inhibitorsoractivatorsaccordingtomanufacturesinstructions.
    3. Add100 μLofFixingSolutionintoeachwellandincubatefor20 minatRTwithshaking.
    4. Add200 μLofprepared1XQuenchingBufferandincubate20 minatRT.
    5. Add200 μLofBlockingSolutionandincubatefor1hat37 °C.
    6. Add50 μLof1Xanti-phospho-proteinspecificantibodyoranti-pan-proteinspecificantibodytoeachwellandincubatefor2hatRT.
    7. Add50 μLofprepared1XHRP-Anti-RabbitorMouseIgGandincubatefor1hatRT.
    8. Add100 μLofTMBOne-StepSubstrateReagenttoeachwell.
    9. Incubate30 minatRT.
    10. Add50 μLofStopSolutiontoeachwell.
    11. Readat450nmimmediately.
    ReagentPreparation

    NOTE:Thawallreagentstoroomtemperatureimmediatelybeforeuse.Ifwashbufferscontainvisiblecrystals,warmtoroomtemperatureandmixgentlyuntildissolved.
    NOTE:Brieflycentrifuge(~1,000g)ITEMSG,H,andIbeforeopeningtoensuremaximumrecovery.
    Formoreinformationlookatthepicture.

    AssayProcedure

    NOTE:ALLincubationsandwashstepsmustbeperformedundergentlerockingorrotation(~1-2cycles/sec).
    1.Designyourexperiment.Forexample,seeFigure2below.
    OPTIONAL:IfseedingHUVECs,HMEC-1orotherlooselyattachedcells,coattheUncoated96-WellMicroplate(ITEMA)byadding100µLpoly-L-Lysine(RecommendedSigmaAldrich)intoeachwellandthenfollowmanufacturersinstructions.Apre-coatedCellBIND®microplateorotherpoly-lysinetreatedtissuecultureplatemaybeusedinplaceofItemA.
    2.Seed100µLof30,000cellsintoeachwelloftheUncoated96-WellMicroplate(ITEMA)providedandincubateovernightat37°Cwith5%CO2.
    NOTE:Theoptimalcellnumberusedwillvaryonthecelllineandtherelativeamountofproteinphosphorylation.Moreorlesscellsmaybeusedbutthismustbedeterminedempirically.
    NOTE:Thecellscanbestarved~4-24hours(dependingoncellline)priortotreatmentwithinhibitorsoractivators.
    3.Applyvarioustreatments,inhibitors(suchassiRNAorchemicals)oractivatorsaccordingtomanufacturersinstructionsandincubateforthedesiredtimepoints.
    NOTE:Itisrecommendedtodissolveinhibitorsoractivatorsintoserum-freecellculturemediumbeforetreatingthecells(unlessotherwisestatedinthemanufacturersinstructions.)
    4.Discardthecellculturemediumbyflippingthemicroplateupsidedownandgentlytappingthebottomofthemicroplateoverasink.
    5.Washbypipetting200µLoftheprepared1XWashBufferA(ITEMB)intoeachwell.Discardthewashbuffer(sameasstep4)andwash2moretimesforatotalof3washesusingfreshwashbuffereachtime.Afterthefinalwash,gentlyblotthemicroplateontoapapertoweltoremoveanyexcess/remainingbuffer.
    NOTE:Toavoidcellloss,donotpipettedirectlyontothecells.Instead,gentlydispensetheliquiddownthewallofcellculturewells.Alsoavoidtheuseofvacuumsuctionortooforcefullytappingthemicroplatewhendiscardinganysolution.
    6.Add100µLofFixingSolution(ITEMD)intoeachwellandincubatefor20minutesatroomtemperature.
    NOTE:Thefixingsolutionisusedtopermeabilizethecells.
    7.Repeatwashstep5.
    8.Add200µLoftheprepared1XQuenchingBuffer(ITEME)intoeachwellandincubate20minutesatroomtemperature.
    NOTE:Thequenchingbufferisusedtominimizethebackgroundresponse.
    9.Wash4timeswith1XWashBufferA.
    10.Add200µLoftheprepared1XBlockingBuffer(ITEMF)intoeachwellandincubatefor1hourat37°C.
    11.Wash3timeswiththeprepared1XWashBufferB(ITEMC).
    NOTE:Ifneeded,themicroplatemaybestoredat-80°Cforseveraldaysafterthiswash.
    12.Add50µLoftheprepared1Xprimaryantibody(ITEMGorH)intoeachcorrespondingwellandincubatefor2hoursatroomtemperature.
    13.Wash4timeswith1XWashBufferB.
    14.Add50µLoftheprepared1XHRPConjugatedsecondaryantibody(ITEMI)intoeachwellandincubatefor1houratroomtemperature.
    15.Wash4timeswith1XWashBufferB.
    16.Add100µLoftheTMBSubstrate(ITEMJ)intoeachwellandincubatefor30minutesatroomtemperatureinthedark.
    17.Add50µLoftheStopSolution(ITEMK)intoeachwell.Readat450nmimmediately.

    RestrictionsForResearchUseonly
    HandlingAdviceAvoidrepeatedfreeze-thawcycles.
    Storage-20°C
    StorageCommentTheentirekitmaybestoredat-20°Cforupto6monthsfromthedateofshipment.Avoidrepeatedfreeze-thawcycles.
    ExpiryDate6months
    SupplierImages
    image for JNK ELISA Kit (Mitogen-Activated Protein Kinase 8) (ABIN1981833)Cell-Basedproteinphosphorylationprocedure
    image for JNK ELISA Kit (Mitogen-Activated Protein Kinase 8) (ABIN1981833)Thispictureshowsthereagentpreparation.
    image for JNK ELISA Kit (Mitogen-Activated Protein Kinase 8) (ABIN1981833)Exampleofhowtoseedcellsforcell-basedassay
    image for JNK ELISA Kit (Mitogen-Activated Protein Kinase 8) (ABIN1981833)Helacellswerestimulatedbydifferentconcentrationsofanisomycinfor15minutesa...
    image for JNK ELISA Kit (Mitogen-Activated Protein Kinase 8) (ABIN1981833)Helacellswerestimulatedbydifferentconcentrationsofanisomycinfor1hourat37°C
    Western Blotting (WB) image for JNK ELISA Kit (Mitogen-Activated Protein Kinase 8) (ABIN1981833)
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