| Assay Method Information | |
| | KLHDC2 Binding Assay |
| Description: | The following is a description of an assay for evaluating ligand binding to KLHDC2.Amplified Luminescence Proximity Homogenous Assay (AlphaScreen) was used to monitor protein-protein interaction of two tagged components that are immobilized on beads. One component was GST-KLHDC2 (E3) and the other one was biotinylated-SelK peptide with a length of 12 amino acids (Substrate peptide). When the two components bind, they bring the Alpha beads in close proximity to each other that results in a luminescent signal. A compound that has affinity for KLHDC2 will compete with the biotinylated-SelK peptide, preventing the beads from being in proximity to each other, therefore, reducing the luminescent signal in a dose dependent manner. The effects of DMSO on the AlphaScreen readout were tested and it was established that this organic solvent used to dissolve most of the hit compounds has detectable but marginal effects on the assay. When DMSO was kept below 5%, its effect on the AlphaScreen assay was negligible.AlphaScreen assays for determining and measuring protein-protein interactions were performed using EnSpire reader (PerkinElmer). GST-tagged KLHDC2 was attached to anti-GST AlphaScreen acceptor beads. Synthetic biotinylated 12 aa SelK degron peptide (Bio-Synthesis, Inc.) was immobilized to streptavidin-coated AlphaScreen donor beads. The donor and acceptor beads were brought into proximity by the interactions between the SelK peptide and KLHDC2. Excitation of the donor beads by a laser beam of 680 nm promotes the formation of singlet oxygen. When an acceptor bead is in close proximity, the singlet oxygen reacts with thioxene derivatives in the acceptor beads and causes the emission of 520-620 nm photons, which are detected as the binding signal. If the beads are not in close proximity to each other, the oxygen will return to its ground state and the acceptor beads will not emit light. Competition assays were performed in the presence of representative binding compounds, which were titrated at various concentrations. |
| Affinity data for this assay | |
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