Specifications

LifeSensors’diubiquitinsubstratesrepresentanewclassofsubstratesforthecontinuousfluorescentmeasurementoftrueisopeptidaseactivity.TheC-terminusofwildtypeubiquitinisconjugatedviaanisopeptidebondtolysine63(K63)ofasecondubiquitinmoleculewiththeresultantdiubiquitinforminganinternallyquenchedfluorescentFRETpair(IQF).Eachubiquitinislabeledwithasinglemoleculeofeitherafluorescentreporter(i.e.TAMRA)orahighlyefficientquenchingfluorophore.CleavageoftheIQFDiUbbydeubiquitylasesleadstoseparationofthefluorophorefromquencherandsubsequentincreaseinobservedfluorescence.

IQFDiUbVariantsThereisaremarkabledifferencebetweenK48-andK63-linkedubiquitinmoleculesintheirthree-dimensionalstructure,whichaccountsfortheirindividualfunctions.Beyonddifferentiallinkages(e.g.K48,K63,K11),LifeSensorshascreatedsubpanelsofIQFdiubiquitinsubstrateswithineachlinkage.BecauseeachDUBislikelytorecognizeandcleavesubstrateswithuniquestericconsiderations,thesesubpanelsvaryinlocationofreporterfluorophoreandquencher.ItisrecommendedthateachDUBbeempiricallyevaluatedagainstapanelofIQFDiUbsubstratestoselectthetheoptimalfluorophore/quencherpairing.Inordertorapidlydeterminewhichofthesefluorophore/quencherpairsareoptimalforyourDUBand/ordeterminedlikagespecificityforyourDUB,LifeSensorsoffersapanelof6substratescomprisedofeachK63-linkedsubstrateavailabletodate.Needbulkquantities?Wewouldbehappytoworkoutacustompricethatfitsyourbudget.Pleaseemailinfo@lifesensors.comformoreinformation.ThisproductiscoveredbyUSpatent#8,518,660.Bypurchasingthisproduct,thepurchaseragreestocomplywiththetermsofourLimitedUseLabelLicense.

Info

KitComponents:-DiUb63-1-DiUB63-2-DiUB63-3-DiUB63-4-DiUB63-5-DiUB63-6AdditionalMaterialsRequired(seemanualformoreinformation):-384-well (GreinerBioONE781209) or96-wellassayplates (GreinerBioOne655076),black-Fluorescentplatereader-FiltersormonochromatorscompatIBLewithmonitoringthefluorescenceofTAMRA(Exc.540nm/Emm.580nm)arerequired-Useofadichroicmirrorwithacutoffintherangeof550-570isrecommendedtoensuretheoptimalsignal-to-background-ControlEnzyme(USP2core)

Amount	Allsubstratesare25µLof20µM
State	Liquid
Linkage	K63
Buffer	50mMSodiumMES,pH6.0
Variants	K63-(1-6)
Fluorescentreadout	TAMRA
Storage	IQFDiUbsarestablefor4-6monthsat4ºC.Longterm(>6months)storageat-80ºCisrecommended.Avoidrepeatedfreeze/thawcycles.

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。