| Assay Method Information | |
| | Homogeneous Time Resolved Fluorescence (HTRF) |
| Description: | DCAF15 interactions with test compounds were monitored using HTRF competition assays (Cisbio, Bedford, MA). Test compounds were first collected in a 384-well microplate (cat. #781280, Grenier Bio-One, Monroe, NC) as 10 mM stocks in 100% dimethyl sulfoxide (DMSO). A second stock plate (cat. #781280, Grenier Bio-One, Monroe, NC) was prepared through 3-fold serial dilution in DMSO of the afore-mentioned stocks using a Mosquito HTS dispenser (mosquito@ HTS, SPT Labtech, Boston, MA). 0.2 μL of these diluted stocks were then dispensed into a 384-well Optiplate (cat. #6007290, Perkin Elmer, Waltham, MA) in duplicate wells, to assemble the assay plate. The tracer molecule containing an Alexa Fluor 647 probe was prepared in 25 mM HEPES pH 7.5, 100 mM NaCl, 0.1 mg/ml BSA, 0.005% Tween 20, 0.5 mM TCEP (dilution buffer) to 612 nM (2.04×). Separately, His-tagged DCAF15 complex was prepared at 32 nM (4×) in the dilution buffer, and mAb Anti-6His-Eu cryptate Gold (cat. #61H12KLA, Cisbio, Bedford, MA) at 4× dilution in the detection buffer (cat. #61DB9RDF, Cisbio, Bedford, MA). These two solutions were mixed and incubated for 15 min at room temperature. 9.8 μL of the tracer solution and 10 μL of the DCAF15/mAb Anti-6His-Eu cryptate Gold mixture were added sequentially to each well in the assay plate. Reaction mixtures with no DCAF15 added were included as positive controls. The final mixture was incubated for an hour at room temperature and spun down briefly before data collection at 615 nm and 666 nm using EnVision 2104 Multilabel Plate Reader (Perkin Elmer, Bedford, MA). |
| Affinity data for this assay | |
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