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,pH7.2,0.05%sodiumazide

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 

 Applications

•      Convenientone-steppulldownofpolyubiquitinatedproteinfromcellandtissueextracts.
•      Isolateubiquitinatedproteinsforproteomicstudies
•      Confirmationoftargetproteinubiquitylation

• Manual

 Benefits         

•      Upto1000-foldhigheraffinityforpolyubiquitincomparedtosingleUBA
•      Avoidsoverexpressionofepitope-taggedubiquitinforpulldowns
•      Protectspolyubiquitinatedproteinsfromdegradationduringcelllysisandsubsequentpulldown
•      Unambiguousdeterminationoftheubiquitylationstatusoftargetproteins
•      Canbemultiplexedtoexamineseveraldifferenttargetsinasinglesample
•      Facilitatesquantitationofpercentubiquitylation
•      Magneticbeadsmakethepulldownefficientandminimizenonspecificbinding

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