AlphaScreen FITC Detection Kit, 500 Assay Points

This kit contains enough reagents to run 500 wells in 384-well format, using a 25 µL reaction volume. This bead kit can be used for many applications, including:

• Protein-protein interaction assays
• Protein-peptide interaction assays
• DNA-protein interaction assays
• RNA-protein interaction assays
• FITC tagged protein detection and quantitation
• Protein expression
• Binding studies

In a typical AlphaScreen assay, one biomolecule of interested is biotinylated and associates with the Streptavidin-coated Donor beads. The other biomolecule is FITC-tagged, and associates with the AlphaScreen anti-FITC Acceptor beads. If the two biomolecules bind to each other, the Donor and Acceptor beads are brought into proximity. Excitation of the Donor beads causes the release of singlet oxygen, which diffuses and triggers the emission of light from the Acceptor beads when in proximity. The amount of light is directly proportional to the degree of interaction. Competition formats are also possible, using the biotinylated FITC probe provided in the kit.

AlphaScreen® and AlphaLISA™ are bead-based assay technologies used to study biomolecular interactions in a microplate format. The acronym ""Alpha"" stands for amplified luminescent proximity homogeneous assay. As the name implies, some of the key features of these technologies are that they are non-radioactive, homogeneous proximity assays. Binding of molecules captured on the beads leads to an energy transfer from one bead to the other, ultimately producing a luminescent/fluorescent signal. To understand how a signal is produced, one must begin with an understanding of the beads. AlphaScreen and AlphaLISA assays require two bead types: Donor beads and Acceptor beads. Each bead type contains a different proprietary mixture of chemicals, which are key elements of the AlphaScreen technology. Donor beads contain a photosensitizer, phthalocyanine, which converts ambient oxygen to an excited and reactive form of O2, singlet oxygen, upon illumination at 680 nm. Please note that singlet oxygen is not a radical; it is molecular oxygen with a single excited electron. Like other excited molecules, singlet oxygen has a limited lifetime prior to falling back to ground state. Within its 4 µsec half-life, singlet oxygen can diffuse approximately 200 nm in solution. If an Acceptor bead is within that proximity, energy is transferred from the singlet oxygen to thioxene derivatives within the Acceptor bead, subsequently culminating in light production at 520-620 nm (AlphaScreen) or at 615 nm (AlphaLISA). In the absence of an Acceptor bead, singlet oxygen falls to ground state and no signal is produced. This proximity-dependent chemical energy transfer is the basis for AlphaScreen's homogeneous nature.