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AlphaLISA® Acceptor beads designed to detect human Histone H3 that is unmodified at lysine 9 and lysine 27 in a homogeneous AlphaLISA assay.
In the AlphaLISA assay, a biotinylated Histone H3 peptide substrate is used in a biochemical enzymatic reaction with histone deacetylase (HDAC) enzymes. When the enzymatic reaction is stopped, the level of deacetylation of lysine 9 or lysine 27 on Histone H3 (depending on substrate design) is determined using antibody-coated AlphaLISA Acceptor beads and streptavidin-coated Alpha Donor beads. The streptavidin Donor beads capture the biotin moeity on the peptide substrate, while the antibody on the AlphaLISA Acceptor beads recognizes unmodified H3K9 or H3K27. Upon laser irradiation of the bead complexes at 680 nm, short-lived singlet oxygen molecules produced by the Donor beads can reach the Acceptor beads in proximity to generate an amplified chemiluminescent signal at 615 nm. The intensity of light emission is proportional to the level of substrate modification.
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.
|Bead Type or Core Bead Type||AlphaLISA Acceptor|
|Experimental Type||In vitro|
|Product Brand Name||AlphaLISA|
|Shipping Condition||Blue Ice|
|Unit Size||5 mg|
Epigenetics, the study of changes in gene expression, is a rapidly growing field which, presents a significant opportunity for drug discovery. As the understanding of the role, of epigenetic changes in various disease states advances, the need for high quality, reagents that can rapidly identify and optimize therapeutic molecules that target these, enzymes is increasing.
AlphaLISA technology is a powerful and versatile platform that offers, highly sensitive, no-wash immunoassays using Alpha Donor and, AlphaLISA Acceptor beads. In this technical note, we present the, optimization of an HDAC1 enzymatic assay using a biotinylated, histone H3K9ac peptide as substrate. Detection of the deacetylated, product was performed by the addition of Streptavidin (SA) Alpha, Donor beads and AlphaLISA Acceptor beads conjugated to an antibody, (Ab) directed against the unmodified H3K9 residue. Upon laser, irradiation of the beads-target complexes at 680 nm, short-lived, singlet oxygen molecules produced by the Donor beads can reach the, Acceptor beads in proximity to generate an amplified chemiluminescent, signal at 615 nm. The intensity of the light emission is proportional to, the deacetylation activity of the HDAC1 enzyme.
This AlphaLISA immunodetection assay measures the demethylation, of a biotinylated histone H3 (1-21) peptide mono-methylated at, lysine 9.