Revvity, AL725F, AlphaLISA Human Total Histone H2A Detection Kit, 5,000 Assay Points

Histone H2A is one of the five main histone proteins involved in the structure of chromatin in eukaryotic cells. Histones H2A, H2B, H3 and H4 are known as the core histones, while histones H1 is known as the linker histone. Two of each of the core histones assemble to form one octameric nucleosome core and the linker histone H1 binds the nucleosome at the entry and exit sites of the DNA. The nucleosome core is formed of two H2A-H2B dimers and a H3-H4 tetramer. H2A is important for packaging DNA into chromatin. Since H2A packages DNA molecules into chromatin, the packaging process will affect gene expression. H2A plays a major role in determining the overall structure of chromatin and regulating gene expression. The AlphaLISA detection of epigenetic marks in cellular extracts is performed as follows: cells 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. The AlphaLISA anti-H2A (C-terminus) Acceptor beads and Biotinylated anti-Histone H2A (epitope near T77) antibodies are then added for the capture of histone proteins. 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.