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
Documents
Benefits
- Upto1000-foldhigheraffinityforpolyubiquitincomparedtosingleUBA
- Avoidsoverexpressionofepitope-taggedubiquitinforpulldowns
- Protectspolyubiquitinatedproteinsfromdegradationduringcelllysisandsubsequentpulldown
- Unambiguousdeterminationoftheubiquitylationstatusoftargetproteins
- Canbemultiplexedtoexamineseveraldifferenttargetsinasinglesample
- Facilitatesquantitationofpercentubiquitylation
- Magneticbeadsmakethepulldownefficientandminimizenonspecificbinding
Life Sensors的泛素和泛素链泛素是一种小的多肽，通过其C端与目标蛋白上的赖氨酸的ε-氨基缀合。这种缀合称为单泛素化。随后，可以利用遍在蛋白表面上的七个赖氨酸残基（K6，K11，K27，K29，K33，K48，K63）中的任何一个，将额外的遍在蛋白部分与初始遍在蛋白结合。这种泛素链形成称为多泛素化。泛素的酶结合是通过一系列酶进行的，即泛素激活酶E1，泛素结合酶E2和泛素连接酶E3。蛋白质的泛素化在细胞中是可逆的。单泛素化和多泛素化的链均被去泛素化酶催化的水解作用裂解 （DUB）。LifeSensors在遍在泛素途径的所有水平的酶（包括E1，E2和E3酶和DUB）上都有丰富的经验。LifeSensors对蛋白质纯化的广泛了解使我们能够生产大约30种E2酶，20种E3酶和35个DUB，并具有定制表达的能力。此外，我们广泛的检测方法简化了确认酶活性和确定E2-E3对兼容性的过程，并确保您可以选择最能解决希望探索的特定，关键生物学问题的酶。LifeSensors已为您的研究开发了多种泛素衍生物，包括用于结合相互作用研究的DUB耐药性聚泛素链，用于DUB分析和筛选的泛素结合物（荧光团和报告酶），选择性泛素链（二，三和四环素）泛素链）用于DUB特异性和结合测定，位点特异性赖氨酸泛素突变体用于E3连接酶和DUB选择性测定。
