Summary:ImmunogenFunctionsasap65decoythroughphosphorylationoftheSer276siteonthepeptide.ModificationpSer276PreparationMethodPreparationof5mMStockSolutions:PBS*isaddeddirectlytothevialstopreparethestocksolutions.Note:Bringthesolutiontoroomtemperatureandquickspinthetubesbeforeopeningthecaps.NF-kBp65(Ser276)InhibitorPeptide:1mgofDRQIKIWFQNRRMKWKKQLRRPSDRELSE)Add52.2ulofPBS*tothevialtomakea5mMstocksolution.Mixbyvortexing.Aliquotandstoreat-20degreeCor-80degreeC.Avoidrepeatedfreezethawing.ControlPeptide:1mgofDRQIKIWFQNRRMKWKKAdd84.8ulPBS*tothevial.Mixbyvortexing.Aliquotandstoreat20degreeCor-80degreeC.Avoidrepeatedfreezethawing.*Recipefor1XPBS:1.Dissolvethefollowingin800mldistilledH2O:-8gofNaCl,0.2gofKCl-1.44gofNa2HPO4-0.24gofKH2PO42.AdjustpHto7.5withHCl.3.Adjustvolumeto1LwithadditionaldistilledH2O.4.SterilizebyautoclavingContentNF-kBp65(Ser276)Inhibitorpeptide(NBP2-31221):2x1mg(lyophilized)DRQIKIWFQNRRMKWKKQLRRPSDRELSE(p65sequence:QLRRPSDRELSE).Molecularweight:3830AntennapediaControlpeptide(NBP2-29334):2x1mg(lyophilized)DRQIKIWFQNRRMKWKK.Molecularweight:2361GeneRELAApplications/Dilutions:ApplicationNotesApplication:InhibitionofDNAbindingactivityofNF-kB.Researcherscanstudytheeffectofp65inhibitorpeptideusingavarietyofmethods.Thisisageneralprotocolandmayneedtobeoptimized.Preincubatecellswithappropriateamountsofinhibitoryorcontrolpeptidesfor1hrandthentreatwithTNForotherNF-kBactivatingagents.PreparenuclearextractsandcheckforthepresenceofNF-kBDNA-bindingactivitybyEMSA(Figure1).NuclearextractscanbepreparedasdescribedbyBharatiA,etal.andTakadaetalorusingnuclearextractionkit.PleaserefertoTakadaetal,2004forfurtherdetailsontheuseofthisinhibitorypeptide.UseinFunctionalinhibitionreportedinscientificliterature(PMID24737129)PublicationsReadPublicationsusingNBP2-26505inthefollowingapplications:B/N1publicationFunc1publicationFunc-Inh2publicationsWB1publicationinvitro1publicationReactivityNotes:Broad:Peptidesequenceis100%conservedacrossmultiplespecies.Reactivityincludeshuman,mouse,rat,dog,andcow.Packaging,Storage&Formulations:StorageStoreat-20C.Avoidfreeze-thawcycles.ConcentrationLYOPHReconstitutionInstructionsPleasecontacttechnicalsupportfordetailedreconstitutioninstructions.Notes:MGC131774nfkbinhibitornfkbp65inhibitornfkbp65nfkbinhibitornfkbp65inhibitorNFkBp65NF-kBp65NFKB3NFKB3v-relavianreticuloendotHeliosisviraloncogenehomologA(nuclearfactorofkappalightpolypeptidegeneenhancerinB-cells3(p65))NuclearfactorNF-kappa-Bp65subunitNuclearfactorofkappalightpolypeptidegeneenhancerinB-cells3transcriptionfactorp65p65inhibitorp65p65RelArelainhibitorrelap65RelAv-relreticuloendotheliosisviraloncogenehomologA(avian)v-relreticuloendotheliosisviraloncogenehomologA,nuclearfactorofkappalightpolypeptidegeneenhancerinB-cells3,p65AlternateNamesfor53BP1Antibody:NF-kBhasbeenshowntoregulatetheexpressionofanumberofgeneswhoseproductsareinvolvedininflammation,viralreplication,carcinogenesis,antiapoptosis,invasion,andmetastasis.Specificadhesionmolecules,chemokines,inflammatorycytokines,andcellcycleregulatorygenesareaffected.Thus,agentsthatcansuppressNF-kBactivationhavethepotentialtobetreatmentsforinflammatorydiseasesandcancer.TheSer276siteofp65isphosphorylatedduringNF-kBactivation,allowingp65nucleartranslocation.Thisp65inhibitorypeptidecontainsaSer276sitethatisphosphorylatedduringNF-kBactivation,therebyblockingp65Ser276phosphorylation(Bhartietal,2003;Takadaetal,2004).TheNF-kBp65(Ser276),alsoknownasPTD-p65-P1,inhibitorypeptidealsocontainsaproteintransduction(PTD)sequence(DRQIKIWFQNRRMKWKK)derivedfromantennapediawhichrendersthepeptidecellpermeable(Derossietal,1994).ThecontrolpeptideconsistsofonlythePTDsequence.ThePTD-p65-P1peptidecaninhibitbindingofrecombinantp65proteintotheDNAinadosedependentmannerandmaximuminhibitionoccursat50uM(Figure1).ItcanalsoinhibitTNF-inducedNF-kBactivationinvivo.PTD-p65-P1suppressesTNF-inducedNF-kBactivationby25%at100uMandcompletelyat150uM(figure2).Background:Thisproductisforresearchuseonlyandisnotapprovedforuseinhumansorinclinicaldiagnosis.Inhibitorsareguaranteedfor1yearfromdateofreceipt.Limitations:NF-kBhasbeenshowntoregulatetheexpressionofanumberofgeneswhoseproductsareinvolvedininflammation,viralreplication,carcinogenesis,antiapoptosis,invasion,andmetastasis.Specificadhesionmolecules,chemokines,inflammatorycytokines,andcellcycleregulatorygenesareaffected.Thus,agentsthatcansuppressNF-kBactivationhavethepotentialtobetreatmentsforinflammatorydiseasesandcancer.TheSer276siteofp65isphosphorylatedduringNF-kBactivation,allowingp65nucleartranslocation.Thisp65inhibitorypeptidecontainsaSer276sitethatisphosphorylatedduringNF-kBactivation,therebyblockingp65Ser276phosphorylation(Bhartietal,2003;Takadaetal,2004).TheNF-kBp65(Ser276),alsoknownasPTD-p65-P1,inhibitorypeptidealsocontainsaproteintransduction(PTD)sequence(DRQIKIWFQNRRMKWKK)derivedfromantennapediawhichrendersthepeptidecellpermeable(Derossietal,1994).ThecontrolpeptideconsistsofonlythePTDsequence.ThePTD-p65-P1peptidecaninhibitbindingofrecombinantp65proteintotheDNAinadosedependentmannerandmaximuminhibitionoccursat50uM(Figure1).ItcanalsoinhibitTNF-inducedNF-kBactivationinvivo.PTD-p65-P1suppressesTNF-inducedNF-kBactivationby25%at100uMandcompletelyat150uM(figure2).

 

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