分类简介：AlphaLISA technology is a no-wash homogeneous proximity immunoassay that includes Alpha Donor and AlphaLISA Acceptor beads to quantitate activity on histone, peptide, or protein substrates. In vitro methylation or acetylation of substrate is performed by incubating enzyme (for example, a histone acetyltransferase or methyltransferase) with S-adenosylmethionine (SAM) or Acetyl CoA cofactor and histone substrate. Following the enzymatic reaction, methylation or acetylation is monitored using mark-specific anti-methyl or anti-acetyl antibody-conjugated AlphaLISA Acceptor beads. Once streptavidin-coated Donor beads are added to capture the substrate, the beads are brought into close proximity. The Donor beads are excited by a laser at 680 nm to release singlet oxygen. The singlet oxygen causes a reaction in the Acceptor bead and light emission is produced at 615 nm. This emission is proportional to the amount of methylated or acetylated substrate present.
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.
Simple assay protocol amenable to automation, eliminating tedious ELISAs and Westerns
Suitable for endogenous and recombinant cell lines, providing the flexibility to work with relevant cell models
Ideal for HTS protocols with demonstrated Z’ factors greater than 0.6
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.
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.
Simple assay protocol amenable to automation, eliminating tedious ELISAs and Westerns
Suitable for endogenous and recombinant cell lines, providing the flexibility to work with relevant cell models
Ideal for HTS protocols with demonstrated Z’ factors greater than 0.6
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.
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.
Simple assay protocol amenable to automation, eliminating tedious ELISAs and Westerns
Suitable for endogenous and recombinant cell lines, providing the flexibility to work with relevant cell models
Ideal for HTS protocols with demonstrated Z’ factors greater than 0.6
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.
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