Md. Boisclair et al., Development of a ubiquitin transfer assay for high throughput screening byfluorescence resonance energy transfer, J BIOMOL SC, 5(5), 2000, pp. 319-328
An assay based on fluorescence resonance energy transfer (FRET) has been de
veloped to screen for ubiquitination inhibitors. The assay measures the tra
nsfer 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 (ubiquiti
n 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 AP
C, 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 di
ssociation-enhanced lanthanide fluoroimmunoassay (DELFIA), However, compare
d to the DELFIA method, use of FRET resulted in higher precision (4% vs. 11
% intraplate coefficient of variation). Quenching of fluorescence was minim
al when compounds were screened at 10 mug/ml using FRET. Employing a quick
and simple homogeneous method, the FRET assay for ubiquitin transfer is ide
ally suited for high throughput screening.