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HECT E3 ligase reacts with UbFluor. This initial transthiolation (k1) results in a HECT E3~Ub thioester and the release of FluorSH. HECT E3~Ub is subsequently cleared by isopeptide bond ligation, regenerating free HECT E3. Thus, defects in transthiolation or isopeptide ligation will both be detected by the UbFluor assay MT reaction conditions, in which there is excess of UbFluor over enzyme.
