AlphaLISA™ Acceptor beads designed to detect human Histone H3 that is mono- and di-methylated at lysine 27 (H3K27me2-1) in a homogeneous AlphaLISA assay. Broad species cross-reactivity is expected based on sequence similarity. Source of antibody: monoclonal.

The anti-methyl-Histone H3 Lysine 27 (H3K27me2-1) AlphaLISA Acceptor beads were used for the development and optimization of an EZH2 methyltransferase assay using a biotinylated Histone H3 (21-44) as substrate. A JMJD3 demethylase assay was also developed, using a biotinylated Histone H3 (21-44) peptide trimethylated at lysine 27 as substrate. Technical notes describing both assays are available in our product literature.

Features:

• No-wash epigenetic assay
• Fully-validated mark specificity
• Substrate flexibility (peptide, protein, histone, nucleosome substrates)
• Easy-to-automate
• Fast assay optimization

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.