Abstract
The DARET (Depolarization After Resonance Energy Transfer) assay is a homogenous, coupled FRET-fluorescence polarization (FRET-FP) assay for BoNT/A or /E proteolytic activity that relies on a fully recombinant substrate. The substrate consists of BFP and GFP proteins flanking SNAP-25 residues 134–206. In the assay, the substrate is excited with plane-polarized light at 379 nm, which primarily excites the BFP, while emission from the GFP at 509 nm is monitored. Energy transfer from BFP to GFP in the intact substrate results in a substantial depolarization of the GFP emission. Upon cleavage by toxin, the BFP and GFP domains are spatially separated, the energy transfer is eliminated, and the polarization of the GFP emission increases. This increase in polari¬zation can be monitored to assay the proteolytic activity of low picomolar concentrations of Type A and Type E toxin in real time. The assay is amenable to high-throughput applications, and the substrate can also be used in a traditional FRET assay.
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