Development of a ubiquitin transfer assay for high throughput screening by fluorescence resonance energy transfer

An assay based on fluorescence resonance energy transfer (FRET) has been developed to screen for ubiquitination inhibitors. The assay measures the transfer of ubiquitin from Ubc4 to HECT protein Rsc 1083, Secondary reagents (streptavidin and antibody to glutathione-S-transferase [GST]), pre-labeled with fluorophores (europium chelate, Eu3+, and allophycocyanin [APC]), are noncovalently attached via tags (biotin and GST) to the reactants (ubiquitin and Rsc), When Rsc is ubiquitinated, EU3+, and APC are brought into close proximity, permitting energy transfer between the two fluorescent labels. FRET was measured as time-resolved fluorescence at the emission wavelength of APC, almost entirely free of nonspecific fluorescence from En(3+) and APC, The FRET assay generated a lower ratio of signal to background (8 vs. 31) than an assay for the same ubiquitination step that was developed as a dissociation-enhanced lanthanide fluoroimmunoassay (DELFIA), However, compared to the DELFIA method, use of FRET resulted in higher precision (4% vs. 11% intraplate coefficient of variation). Quenching of fluorescence was minimal when compounds were screened at 10 mug/ml using FRET. Employing a quick and simple homogeneous method, the FRET assay for ubiquitin transfer is ideally suited for high throughput screening.