Reader Domain Assay Services

Reaction Biology offers biochemical and biophysical assays to study epigenetic reader domains. More than 100 assays are available for screening, lead optimization, or selectivity profiling for reader domain inhibitors.

Several potent reader domain compounds have recently been identified, increasing the appreciation of this family’s functional importance and therapeutic potential. At Reaction Biology, we are continuously developing new reader domain assays.

  • Extensive coverage of the bromodomain family.
  • All reader domain proteins are produced at our facility and are available for purchase.
  • The BromoMELTTM assay kit is available for in-house screening.

The Largest Bromodomain Assay Panel in Industry

Bromodomain proteins are epigenetic readers that bind to acetylated lysine residues on histones and regulate gene transcription. The phylogenetic tree shows the relationships between genes predicted to encode for bromodomain proteins.

all bromodomain containing epigenetic targets shown as a phylogenetic treee

Assay Details

Assay format: AlphaScreen

AlphaScreen assays stand for amplified luminescent proximity homogeneous assay.

assay principle alphascreen for bromodomain screening of compounds

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.

Assay format: Thermal Shift

 

thermal shift assay principle and example graph

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.

 

 

thermal shift assay with JQ1 targeting BRD2 bromodomain

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.

Assay format: HTRF

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 principle for compound screening of bromodomains

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.

Assay setup

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

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, % enzyme activity 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.

Selectivity Profiling

For more information about the selectivity screening of bromodomain proteins visit our BromoMELTTM Assay Kit page