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The AlphaLISA® Epigenetic Cellular Detection Kits enable rapid and direct detection of endogenous modification of epigenetic marks on histone H3. These no wash, all-in-one-well assays are optimized for simplicity and throughput.
The AlphaLISA detection of epigenetic marks in cellular extracts is performed as follows: cell cultured in the presence of compounds are lysed with the Cell-Histone Lysis buffer. Histones are then extracted from the nucleosomes by the addition of the Cell-Histone Extraction buffer. AlphaLISA anti-mark Acceptor beads and Biotinylated anti-Histone H3 (C-terminus) antibodies are then added for the capture of histone proteins carrying the mark of interest. After incubation, Streptavidin Donor beads are added for the capture of the biotinylated antibody. In the presence of histone proteins bearing the mark of interest, the beads come into proximity. Excitation of the Donor beads provokes the release of singlet oxygen molecules that trigger a cascade of energy transfer reactions in the Acceptor beads, resulting in a sharp peak of light emission at 615 nm.
|Assay Target||Histone H3|
|Assay Target Class||Histone|
|Experimental Type||In vitro|
|Product Brand Name||AlphaLISA|
|Shipping Condition||Blue Ice|
|Unit Size||5,000 assay points|
The AlphaLISA technology allows performing no-wash homogeneous, proximity immunoassays using Alpha Donor and AlphaLISA, Acceptor beads. In this technical note, we present an optimized, assay for measuring changes in the levels of H3K27ac after treatment, of cells with sodium butyrate and Trichostatin A (TSA), two nonselective, histone deacetylase (HDAC) inhibitors. Following a, homogeneous histone extraction protocol, the mark of interest, is detected by the addition of a biotinylated anti-Histone H3, (C-terminus) antibody and AlphaLISA Acceptor beads conjugated, to an antibody (Ab) specific to the mark. The biotinylated antibody is, then captured by Streptavidin (SA) Donor beads, bringing the two, beads into proximity. Upon laser irradiation of the Donor beads, 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.