LanthaScreen conjugation assay reagents provide sensitive high-throughput screening (HTS) reagents to either monitor the rate of conjugation of ubiquitin or ubiquitin-like (UBL) molecules to target proteins, or the extent of this conjugation.  By incorporating the TR-FRET donor (terbium) or acceptor (fluorescein) onto ubiquitin and SUMO proteins, these universal assay reagents can be used to rapidly develop screening assays for conjugating enzymes.  To maintain biochemical reactivity, the ubiquitin and UBL conjugates are selectively modified near the N-terminus via an introduced cysteine residue with thiol reactive dyes.  This type of chemical modification leaves all of the internal lysine residues free for conjugation to another ubiquitin or UBL molecule to form poly-ubiquitin (or UBL) chains.

For a typical HTS TR-FRET ubiquitination assay, fluorescein- and/or Terbium-labeled ubiquitin is incubated with the ubiquitin-conjugating enzymes (E1, E2 and E3), a target protein to be ubiquitinated, and an ATP solution to initiate the reaction.  The enzymes conjugate the labeled ubiquitins to the target protein, resulting in mono- or poly- ubiquitination.  Depending on the specific assay, a detection reagent (a Tb-anti-epitope-tagged antibody) may be added to the reaction to complete the TR-FRET pairing (Figures 1 and 2).  Similar assays reagents have also been developed for the Ubiquitin-like proteins SUMO1, SUMO2, and SUMO3.

Assay schematics for the ubiquitination of an epitope-tagged protein

Figure 1. Assay schematics for the ubiquitination of an epitope-tagged protein.

 

Assay schematic and representative data for poly-chain ubiquitination assay

Figure 2.  Assay schematic and representative data for poly-chain ubiquitination assay. When an epitope tag is not available on the target protein, or when real-time polyubiquitination detection is desired, the intrachain (A) ubiquitination assay is used. Since both the TR-FRET donor (Tb–ubiquitin) and acceptor (fluorescein–ubiquitin) are present in the poly-ubiquitin chain, no development step is required for the intrachain assay. This makes the intrachain assay especially useful when real-time kinetics information on ubiquitination is required. Representative data from an intrachain ubiquitination assay using the enzyme UbcH1 illustrates the high TR-FRET values achieved with this assay as compared to controls (B), competition of methylated ubiquitin with the Tb–ubiquitin and fluorescein–ubiquitin to form polyubiquitin chains (C), and a robust Z’-factor of 0.92 representative of data achieved with this assay (D). The elimination of the development step for this assay allows the ubiquitination reaction to be monitored in real time to provide valuable kinetic information (E).

 

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