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.
Example of HTRF assay principle for screening compounds inhibiting the binding of BCL-2 to Bak. Antibodies recognizing the GST tagged-BCL-2 protein are coupled with the donor fluorophore (Terbium-chelate). The Bak peptide substrate is coupled to an acceptor fluorophore (FAM). The binding of BCL-2 to Bak cause a proximity of acceptor and donor fluorophore enabling FRET upon excitation.
The fluorescence polarization assay is based on the principle that the degree of excitation by polarized light of a small fluorescently labeled peptide changes with the degree of rotation. The change of motion after binding to a larger protein is the basis for this homogenous and reproducible competition binding assay.
Example of fluorescence polarization assay principle for screening compounds targeting cIAP. A substrate peptide binding to the BIR3 domain of cIAP coupled to FAM fluorophore is used as tracer. The fluorescence polarization signal decreases with the addition of compound that interrupts cIAP interaction to the fluorescent substrate.
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.