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 for enhanced assay performance compared to conventional fluorescent probes in terms of sensitivity, assay window, and robustness.
Example of KDM4A assay procedure with HTRF. Enzymatic demethylation of the biotinylated histone H3 lysine 36 residue is detected by a Europium cryptate-labeled antibody specific to the histone methylation status (M2). The histone substrate peptide is biotinylated and binds to streptavidin carrying the acceptor conjugate XL665.
KDM4A was inhibited by reference compounds 2,4-PDCA and IOX1. 2,4 PDCA (IC50 = 138 nM) does inhibit the enzyme 15 x more than IOX1 (IC50 = 2014 nM). Shown is a concentration-response curve with 10 inhibitor concentrations performed in duplicates.
The Amplex Red reagent is a fluorogenic substrate for horseradish peroxidase for quantification of the activity of enzymes that produce peroxide, such as demethylase LSD1. The Amplex Red reagent reacts with peroxide in a 1:1 stoichiometry to produce the red-fluorescent oxidation product, resorufin, which can be quantified by a luminometer.
AlphaLISA assays stand for amplified luminescent proximity homogeneous assay.
Demethylase assay procedure with AlphaLISA for example KDM4D. Enzymatic demethylation of the biotinylated histone H3 lysine 9 residue is detected by antibodies specific to the histone methylation status (M2) which are coupled to AlphaLISA acceptor beads. Streptavidin covered donor beads interact with the histone substrate peptide via the biotin tag. The proximity of donor and acceptor beads enables singlet oxygens, a radical oxygen form, to transfer energy to the acceptor beads inducing fluorescence.
Example data of KDM4D inhibited by reference compounds 2,4-PDCA (IC50 = 331 nM). Shown is a concentration-response curve with 10 inhibitor concentrations performed in duplicates.
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, 20ul of a 10mM 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.