AsinglebiotinmoietyisconjugatedontheN-terminusofubiquitin.Alllysineresiduesofubiquitinarestillavailableforformationofpolyubiquitinchains.TheextremelyhighbindingaffinitybetweenbiotinandstreptavidinallowstheuseofN-terminalbiotin-ubiquitinforthefollowinginvitroassays:1)rapidandefficientpurificationofpolyubiquitinatedsubstrateproteins;2)monitoringsubstrateubiquitinationusinganHRP-conjugatedstreptavidinantibody;and3)settinguphighthroughputFRETassaystomonitorsubstrateubiquitinationinassaysscreeningE3ubiquitinligaseinhibitors.
UseDependingonubiquitinationassayconditions,N-terminalbiotin-ubiquitincanusedinconjunctionwithwildtypeubiquitinatcombinedconcentrationrangeof20-50μMwitha1:4to1:20ratioofN-terminalbiotin-Ub:wildtypeubiquitin.
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LC-MSofN-terminalBiotin-Ubiquitin
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