Specifications
Thiskitallowsyoutodeterminewhetherornotyourtargetproteinisubiquitylated.
Themostcommonmethodtoinvestigateproteinubiquitylationisthroughimmunoprecipitationandimmunoblotanalysis;however,thesubstrateantibodymayinteractdifferentlywithanubiquitylatedsubstrateduetoepitopemasking,reducedaffinity,orchangesinselectivity.Amoredefinitivemethodfordemonstratingproteinubiquitylationistocoupleimmunoprecipitationwithdigestionbyabroadspectrumdeubiquitylasepriortoimmunoblotanalysis. Anincreasedsignalfortheproteinofinterest(POI)afterDUBtreatmentisaclearindicationthattheproteinwasubiquitylatedeveniftherewasnoclearreactivityintheuntreatedsample. ToavoidpotentialproblemsarisingfromchangesinimmunoreactivityofthePOI,itisbesttopull-downtheproteinwithananti-ubiquitinreagent.
WehavebuiltthiskitaroundtheuseofMagneticTandemUbiquitinBindingEntities(TUBEs)whichbindtoallubiquitinchainlinkages.Magnetic-TUBEsareTUBEmoietiesdirectlycoupledtomagneticbeads,fortheidentificationandcharacterizationofpolyubiquitinatedproteinsbywesternblottingand/ordownstreamproteomicstudies.Magnetic-TUBEsfacilitateconvenient“one-step”pull-downofpolyubiquitinatedproteins.
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Components
Tube1—MagneticTUBE1–1mlofslurry,100µlslurryisrecommendedfor1-2mgoftotalcelllysatepulldown.
Buffer:PBS,pH7.2,20%ethanol
Storage:PleasekeepthemagneticTUBE1at4°C.Donotcentrifuge,dryorfreezethebeads
Tube2—Washbuffer–2mL
Tube3—Elutionbuffer–1mL
Tube4—10XNeutralizationbuffer(Addβ-mercaptoethanoltoafinalconcentrationof10mMbeforeuse,e.g.add5µlof1Mβ-mercaptoethanolstocksolution)–0.5mL
Tube5–BroadSpectrumDUB–25µg,10µM,Storage,-80°C
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