TheE1LITE–UBE1ActivityAssayKitisbasedonFRETbetweendonorandacceptorfluorophoresassociatedwiththetwoubiquitinmoleculesboundtotheactivatedUBE1ternarycomplex(seefigurebelow).Theassayemploysstreptavidinterbiumasthedonorfluorophore,whichisdirectedtothecomplexbybiotinylatedubiquitinandfluoresceinlabeledubiquitinastheacceptorfluorophore.Thelong-livedfluorescenceoftheterbiumdonorallowsatime-delayedreADIngoftheFRETsignal,ortime-resolvedFRET(TR-FRET),whichreducesthecontributionofshort-livedbackgroundemissionsresultingfromfrombuffers,proteins,andchemicalcompounds.Theassaycanberunina96-wellor384-wellformatandproducesareproducIBLesignalwithaZ’>0.7.Thekitcontainssufficientreagentsforone1/2-volume96-wellplate.

“ThetimeresolvedFRETassayforE1ternarycomplexformationrepresentsasignificantadvanceinspeedandutilitythatshouldbeparticularlyusefulforthoseinterestedinroutinequalitycontrolmeasurements,highthroughputactivityscreens,orrapidreactionkineticsforwhichisotopicmethodsareprecluded.”

–ArthurL.Haas,Ph.D.RolandCoulsonProfessorandHeadDepartmentofBiochemistryandMolecularBIOLOGyLSUHealthSciencesCenterSchoolofMedicine

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