Both assays rely on the same Donor beads yet use different Acceptor beads (Figure 2). PerkinElmer's Alpha technology includes both AlphaLISA and AlphaScreen assays. This proximity-dependent chemical energy transfer is the basis for AlphaScreen's homogeneous nature.ĪlphaLISA and AlphaScreen: understanding the difference In the absence of an Acceptor bead, singlet oxygen falls to ground state and no signal is produced. 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).
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Within its 4 µsec half-life, singlet oxygen can diffuse approximately 200 nm in solution. Like other excited molecules, singlet oxygen has a limited lifetime prior to falling back to ground state. Please note that singlet oxygen is not a radical it is molecular oxygen with a single excited electron. Donor beads contain a photosensitizer, phthalocyanine, which converts ambient oxygen to an excited and reactive form of O 2, singlet oxygen, upon illumination at 680 nm. Each bead type contains a different proprietary mixture of chemicals, which are key elements of the AlphaScreen technology. AlphaScreen and AlphaLISA assays require two bead types: Donor beads and Acceptor beads. To understand how a signal is produced, one must begin with an understanding of the beads. Binding of molecules captured on the beads leads to an energy transfer from one bead to the other, ultimately producing a luminescent/fluorescent signal.
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As the name implies, some of the key features of these technologies are that they are non-radioactive, homogeneous proximity assays. The acronym "Alpha" stands for amplified luminescent proximity homogeneous assay. Principle of the AlphaLISA assay technologyĪlphaScreen ® and AlphaLISA ® are bead-based assay technologies used to study biomolecular interactions in a microplate format.