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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||500 assay points|
The introduction of enzyme-linked immunosorbent assays (ELISAs) in the early 1970’s offered researchers a non-radiometric immunoassay platform without compromising sensitivity. Over the last 50 years scientists have made huge strides in disease research and drug discovery and a demand for greater assay throughput and sensitivity has evolved. In response, more robust immunoassays have been developed to address some of the limitations of the standard, colorimetric ELISA.
Find out about the most common limitations of traditional ELISAs and how different ELISA alternative technologies address these limitations.
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 H3K9ac after treatment of cells, with sodium butyrate and Trichostatin A (TSA), two non-selective, 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.,