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| | RIPK1 Binding Assay |
| Description: | A fluorescent probe containing an Oregon green fluorophore was synthesized from a compound known to bind to RIPK1.The assay was performed in a buffer containing 50 mM N-2-Hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES) (pH 7.5), 10 mM MgCl2, 1 mM ethylene diamine tetraacetic acid (EDTA), and 0.01% BRIJ-35 (Polyoxyethylene (23) lauryl ether). Test compounds were solvated in dimethyl sulfoxide (DMSO) at final concentrations 4-fold serially diluted from 100 to 0.000095 uM in technical duplicates into an empty 384 well plate. Recombinant RIPK1 (final concentration of 2.5 nM) was added to the wells and incubated with test compound for 1 hour at room temperature. A solution containing the fluorescent probe (final concentration 5 nM) and an LanthaScreen Tb-anti-GST antibody (Thermofisher Scientific; final concentration of 5 nM) was then added to each well, with the Tb-anti-GST antibody binding to the GST portion of the recombinant RIPK1 and providing the corresponding FRET pair to the fluorescent probe. The plate was incubated for 1 hour at room temperature and read on an Envision plate reader using a time resolved FRET protocol. Data was normalized and analyzed using Studies from Dotmatics. |
| Affinity data for this assay | |
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