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EpiCypher

AlphaLISA BROMODOMAIN READER ASSAYS

EpiCypher and PerkinElmer have collaborated to develop a family of AlphaLISA kits to measure the binding of BRD4 and P300 bromodomains to various acetylated histone peptide substrates. These assays are highly sensitive and ideal for high throughput binding inhibitor studies.

Bromodomains (BRDs) are a class of epigenetic reader molecules that translate the acetylation state of histones (and other proteins) into downstream cell-signaling events. Due to their importance in these physiological processes, bromodomains are believed to be involved in many disease states, from cancer and atherosclerosis. BRD4, currently the most studied member of the BET (bromodomain and extra terminal domain) family, contains two tandem bromodomains (BD1 and BD2) and an extraterminal (ET) domain. BRD4 directly maintains the acetylation state of histones and preferentially binds to acetylated H3 and H4. The recent discovery of potent and highly specific BET family inhibitors, such as JQ1, has stimulated intensive research activity, and BRD4 is now considered as an important therapeutic target. This assay can be used to determine the binding of BRD4 with acetylated histone H4 peptide and measure any inhibition of this binding.

AlphaLisa Schematic AlphaLisa BRD4 Assay Data

AlphaLISA® detection of epigenetic marks.
Glutathione (GSH) AlphaLISA Acceptor beads are used to capture the GST-tagged bromodomains and Streptavidin-coated Donor beads capture the biotinylated peptide. Donor and Acceptor beads come into proximity through BRD4 binding to the acetylated histone H4 peptide. Excitation of the Donor beads (680 nm) provokes the release of a singlet oxygen that triggers a cascade of energy transfer reactions in the Acceptor beads, resulting in a sharp peak of light emission at 615 nm.

JQ1 inhibition of BRD4 BD1 and H4 K5Ac binding.
The JQ1 concentration ranges from 3 pM to 10 µM. BRD4 (BD1) and H4 K5Ac are 25 nM. The IC50 value of 149.8 nM was calculated by using nonlinear regression fitting with GraphPad Prism 5.

AlphaLISA BRD4 BD1 Assays AlphaLISA BRD4 BD2 Assays
BRD4 (BD1) and H4 K5Ac Binding Assay Kit
BRD4 (BD1) and H4 K8Ac Binding Assay Kit
BRD4 (BD1) and H4 K12Ac Binding Assay Kit
BRD4 (BD1) and H4 K16Ac Binding Assay Kit
BRD4 (BD1) and H4 K5,12Ac Binding Assay Kit
BRD4 (BD1) and H4 K5,8,12Ac Binding Assay Kit
BRD4 (BD1) and H4 K8,16Ac Binding Assay Kit
BRD4 (BD1) and H4 K5,8,12,16Ac Binding Assay Kit
BRD4 (BD2) and H4 K5Ac Binding Assay Kit
BRD4 (BD2) and H4 K8Ac Binding Assay Kit
BRD4 (BD2) and H4 K12Ac Binding Assay Kit
BRD4 (BD2) and H4 K16Ac Binding Assay Kit
BRD4 (BD2) and H4 K5,12Ac Binding Assay Kit
BRD4 (BD2) and H4 K5,8,12Ac Binding Assay Kit
BRD4 (BD2) and H4 K8,16Ac Binding Assay Kit
BRD4 (BD2) and H4 K5,8,12,16Ac Binding Assay Kit
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