EpiQuik BRD2 Binding Activity/Inhibition Assay Kit (Colorimetric)

The EpiQuik™ BRD2 Binding Activity/Inhibition Assay Kit is a complete set of essential components which enables an experimenter to colorimetrically quantify BRD2 binding activity/inhibition. The kit can be used with purified BRD2 proteins or nuclear extracts from fresh tissues or cultured cells from human and mouse. The EpiQuik™ BRD2 Binding Activity/Inhibition Assay Kit has the following features:

• Fast procedure, which can be finished within 6 hours.
• Innovative colorimetric assay which directly measures BRD2 binding activity in a 96-well plate using a standard microplate reade without the need for special reagents or expensive equipment.
• Both cell/tissue extracts and purified BRD2 protein can be used, which allows for the detection of inhibitory effects of BRD2 inhibitors in vivo and in vitro.
• High sensitivity and specificity – BRD2 binding-specific detection with a detection limit as low as 0.1 ng/well bound BRD2 protein.
• Strip microplate format makes the assay flexible via manual or high throughput analysis.
• BRD2 control is included, which allows the binding activity of BRD2 protein in the sample to be properly quantified.
• Simple, reliable, and consistent assay conditions.

Background Information
Bromodomains (BRDs), which are a diverse family of evolutionarily conserved protein-interaction modules, are found in histone acetyltransferases and other chromatin-associated proteins. More than 60 BRDs have been identified, which in turn cluster into eight families based on structure/sequence similarity. BRDs bind selectively to acetylated lysines, acting as "readers" of the histone code, and have recently been shown to regulate transcription activity; they also contain a druggable binding pocket. Proteins that contain BRD2 have been implicated in the development of a large variety of diseases and inhibitors that target BRDs of the BET (Bromodomains and extra-terminal), which inhibit BRD-mediated protein-protein interaction and have the potential to modulate multiple diseases including inflammation and cancer. 

Principle & Procedure
In an assay with this kit, the unique BRD2 ligand is stably coated onto the strip well. The sample is added into the well and BRD2 proteins contained in the sample bind to the ligand. The bound BRD2 can be recognized with a BRD2-specific antibody and colorimetrically quantified through an ELISA-like reaction. The amount of bound BRD2 is proportional to the intensity of color development. 

Starting Materials & Input Amount
Input materials can be nuclear extracts or purified BRD2 protein. The amount of nuclear extracts for each assay can be 1 µg to 20 µg with an optimized range of 5-10 µg. We recommend using Epigentek's nuclear extraction kit for optimal results. The amount of purified protein can be 10 ng to 500 ng, depending on the purity of the protein. 

Fig. 1. Schematic procedure for the BRD2 Binding Activity/Inhibition Assay Kit (Colorimetric).

Fig. 2. Illustrated standard curve generated with the BRD2 control from the EpiQuik BRD2 Binding Activity/Inhibition Assay Kit (Colorimetric) 

Fig. 3. Nuclear extracts were prepared from HeLa cells and the ODs generated using the EpiQuik BRD2 Binding Activity/Inhibition Assay Kit (Colorimetric) were measured.