Bromodomain Assay Service (Reader Domain Assays)

AlphaScreen assays stand for amplified luminescent proximity homogeneous assay. 

Example of bromodomain BRD-1: The His-tagged bromodomain and the substrate, which is a biotin-tagged acetylated peptide, are each captured on AlphaScreen beads. Binding interaction between the peptide and the bromodomain brings the beads in close proximity. Laser excitation of the donor beads allows for energy transfer by singlet oxygen atoms between the fluorophores.
 

Incubation with test molecules binding to the bromodomain results in a diminished interaction with the biotinylated substrate leading to a decline in light emission. 

Thermal shift assays measure the thermal stability of a target protein and the increase in protein melting temperature upon the binding of a ligand to the protein. Our thermal shift bromodomain assays are available as service and as a kit for in-house selectivity screening.

Differential Scanning Fluorimetry of BRD2-Tndm (His) in the Presence or Absence of JQ1. Thermal denaturation of BRD2-Tndm (His) was detected by increased binding and fluorescence of the dye SYPRO Orange. Addition of the BET bromodomain inhibitor JQ1 stabilizes the protein folding and shifts the melting temperature from 41 to 44 degrees Celsius.

HTRF (Homogeneous Time-Resolved Fluorescence) is an assay technology that combines fluorescence resonance energy transfer technology (FRET) with time-resolved measurement (TR) for measuring molecular interactions in a homogeneous format.

HTRF makes use of rare earth complexes such as Europium or Terbium cryptates. These chelates are very stable under a wide range of chemical conditions, are not subject to photobleaching, and show high kinetic stability. HTRF allows enhanced assay performance compared to conventional fluorescent probes in terms of sensitivity, assay window, and robustness.

HTRF assay procedure with a bromodomain: Acetylated peptides are used as substrates for the bromodomains to bind on. Antibodies recognizing the GST tagged-bromodomain are coupled with the donor fluorophore. The peptide is tagged with biotin and recognized by streptavidin which is coupled with an acceptor fluorophore. The proximity of acceptor and donor fluorophore allows for FRET upon excitation.

Incubation with test molecules binding to the bromodomain results in a diminished interaction with the biotinylated substrate leading to a decline in light emission. 

Setups: Single-dose screening in duplicates or IC50 value determination with 5 or 10 concentrations. Other screening formats are available upon request.

The thermal shift assay format allows a 'yes' or 'no' answer whether the test molecule binds to the reader domain protein. IC50 determination is not possible. 

Controls: No inhibitor (DMSO vehicle) control and for every assay, one target-specific control compound is tested in 10-dose IC50 format. 

Turnaround time: 10 business days for standard projects. Expedited scheduling and data delivery can be arranged prior to the commencement of the studies.

Report: The raw data, % inhibition and control compound IC50 values will be reported in Excel format for single-dose assays. For IC50 orders, raw data, IC50 values, and curve fitting will be delivered in Excel format. Assay conditions, target, and substrate information are available upon request. Requirements for this information should be noted prior to the commencement of the study.

Screening facility: This assay is performed at our screening facility in Malvern, PA, US.

Compound requirements: In brief, for a standard project, 20 µl of a 10 mM DMSO stock or solid material is needed. Less material is needed for large-scale screening. Please refer to our FAQs for information regarding compound preparation and shipping.

On a monthly basis, Reaction Biology performs compound screening on the full bromodomain panel.

Please see the upcoming screening dates here.

For more information about the selectivity screening of bromodomain proteins visit our BromoMELT^TM Assay Kit page

We offer two assay formats for intracellular compound screening on bromodomain targets:

1. Binding of a compound to BET Bromodomain in intact cells based on NanoBRET Intracellular Target Engagement assay technology.

2. Testing the protein-protein interaction of bromodomains with acetylated histones in intact cells based on the NanoBRET Bromodomain/Histone Interaction Assay.

Please see more information on our Cell-based Epigenetic Assays webpage.