BackgroundInformation | testPrinciple TheUPSTATEcolorimetricSTAR(SignalTransductionAssayReaction)ELISAkitisasolidphasesandwichenzymelinkedimmunosorbentassaythatprovidesafast,sensitivemethodtodetectspecificlevelsofsignalingtargetsinwholecellextracts.TheJNK1/2plateiscoatedwithaspecificmousemonoclonalJNK1/2captureantibodyonthemicrowellsofthe96-wellclearplate.SamplelysateorthestandardincludedinthekitareincubatedinthemicrowellsallowingJNK1/2antigentobecapturedintheplatewells.Theplateisthenwashedtoremoveanynon-boundunspecificmaterial.Thewellsarethenincubatedwithaspecificrabbitanti-JNK1/2antibodytodetectthecapturedJNKontheplatewell.TheunbounddetectionantibodyiswashedawayfollowedbyincubationwithanHRP-conjugatedanti-rabbitantibody.Thisallowsforasensitiveenzymaticdetectionofthesample.AftertheadditionofTMBsubstrateandstopsolutiontheabsorbanceismeasuredat450nmusingaplatereader.Theentireassaytakeslessthan5hourstocompletewithminimalhands-ontime.Manyofthereagentsaresuppliedinready-touseformulationsforeaseofuse.Thekitalsoincludesastandardthatisrunasbothapositivecontrolandtodevelopastandardlineofdetection.
JNKBackground JNK(c-JunN-terminalKinase,MAPK8)isamemberoftheMAPkinasefamily.JNKwasoriginallyfoundtobestressactivated,andassucharealsoreferredtoasthestress-relatedMAPK.FouralternativelysplicedtranscriptvariantsencodingdistinctisoformsofJNKhavebeenreported.JNKisregulatedbystressinducingsignalssuchasosmoticstressandUVrADIationaswellasproinflammatorycytokinessuchasIL-1andTNFα.JustasErk(p44/p42,MAPK)isactivatedbyitsupstreamkinaseMEK,JNKisphosphorylatedandactivatedbyMKK4andMKK7onthequintessentialMAPKactivationsequenceofThr-X-Tyr,whereXisaProfortheJNKkinasesonThr183/Tyr185forJNK1andThr221/Tyr223forJNK2.Oncephosphorylatedandactivated,JNKgoesontophosphorylatedownstreamtargetsinboththecytoplasmandthenucleus.JNKservesasatranscriptionalregulatorandisknowntophosphorylateanumberofdownstreamtargets,includingthetranscriptionfactorsc-Jun,ATF-2,andELK-1.C-Jun,amemberoftheAP-1familyoftranscriptionfactors,regulatescytokinegeneexpression.Alongtheselines,JNKinhibitorsmaybeeffectiveinthecontrolofrheumatoidarthritis(RH),anautoimmunediseaseinvolvingincreasedproductionofinflammatorycytokines.InhibitionofJNKsignalingalsoenhanceschemotherapy-inducedinhibitionoftumorcellgrowth,suggestingtheJNKsmayprovideamoleculartargetforthetreatmentofcancer.BothJNKandp38MAPKareoftensimultaneouslyactivatedinresponsetoenvironmentalchanges.Thesetwostresskinasesignalingpathwayshaveevolvedtorelayincreasinglycomplexrangesofenvironmentalstimuli.TheJNKpathwayiscriticallyinvolvedindiabetesandlevelsareabnormallyelevatedinobesity.Thecell-permeableJNKinhibitorypeptidemayhavepromiseasatherapeuticagentfordiabetes. |