Loading patent…
AssigneeBoard of Regents, The University of Texas System
InventorsFrank Herkules, Dmitri Ivanov
Estimating the term…
Certain embodiments are based on the discovery of a method for monitoring formation and dissociation of E2-Ubl conjugates in protein ubiquitination reactions. The methods use the fluorescence signal produced by a FRET-active E2-Ubl conjugate, wherein E2 is covalently labeled with one fluorophore, Ubl is labeled with a second fluorophore, and the two fluorophores form a FRET-active pair. The methods can be used in different assays in which E2-Ubl conjugates are formed or dissociated, for example in assays for monitoring enzymatic activity of E1 ubiquitin activating enzymes or enzymatic activity of E3 ubiquitin ligases. The method allows monitoring formation or/dissociation of E2-Ubl conjugates in real time with high sensitivity using widely available scientific instruments for measuring fluorescence and does not require labor intensive and error prone sample processing and analysis procedures. The method enables measurements of E1 or E3 activity in the high-throughput format.
A FRET-active E2-Ub/Ubl conjugate comprising a ubiquitin (Ub) or a ubiquitin-like protein (Ubl) covalently coupled to an E2 protein, the Ub or Ubl protein being modified with a first fluorophore and the E2 protein being modified with a second fluorophore, wherein the first fluorophore and the second fluorophore can transfer energy from one to another via Fluorescence Resonance Energy Transfer (FRET) when in proximity of each other.
(canceled)
(canceled)
(canceled)
(canceled)
(canceled)
(canceled)
(canceled)
(canceled)
(canceled)
(canceled)
(canceled)
(canceled)
(canceled)
(canceled)
A method for monitoring activity of a E1 ubiquitin activating enzyme, the method comprising:
(canceled)
(canceled)
(canceled)
A kit for synthesis of FRET-active E2-Ub/Ubl conjugates comprising a Ub/Ubl labeled with a first fluorophore, an E2 labeled with a second fluorophore, such that the first and the second fluorophores make a FRET-active pair.
(canceled)
(canceled)
Dependencies are inferred from the claim text.