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Assay Method Information

Assay Name:  AlphaScreen Assay
Description:  The assay uses two types of beads: donor beads are coupled with streptavidin, and acceptor beads are coupled with an anti His-tag antibody. Upon excitation with light of a wavelength of 680 nm the donor beads produce reactive oxygen species that travel in solution only for a limited distance. If the acceptor beads are within that travel distance, it will absorb the reactive oxygen and emit light in the range of 520-620 nm. In our assay we have used biotin-coupled PEX5 peptide (coupling to donor beads) and His-tagged T. brucei PEX14 (coupled to the acceptor beads). Thus, PEX5-PEX14 complex dissociation by a ligand caused decrease in light emission as the donor and acceptor beads were separated. Typically, we used 30 nM PEX5, 30 nM PEX14 and bead concentrations of 5 μg/mL. The PEX5-PEX14 complex was incubated with varying concentrations of inhibitor for 1 hour. Next, the beads were added and the system allowed to equilibrate for another hour. The raw data were fitted to four parameter sigmoidal curves by Origin software and EC50 values calculated. Each measurement was performed in quadruplicate and every experiment was accompanied with blank and reference samples (using non-biotynylated PEX5 peptide as a binding competitor).
Affinity data for this assay
 

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Last update November 1, 2007
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