| Assay Method Information | |
| | In Vitro TR-FRET Ternary - ALK Mutant (Mut) CRBN |
| Description: | A TR-FRET proximity assay was used to measure ternary complex formation induced by test compounds. Compounds dissolved in 100% DMSO were dispensed to a 384-well plate by an SPT Labtech Mosquito LV as duplicate 10-point dilution series to a total volume of 100 nanoliters of DMSO. One column of DMSO only and one column of 3-(3-{N-methyl[(3-phenyl-1,2,4-oxadiazol-5-yl)methyl]amino}-2,5-dioxo-3-pyrrolin-1-yl)-2,6-piperidinedione (final concentration 10 μM) served as negative and positive controls, respectively. To this plate was added 10 microliters of a reaction mixture containing 150 nM avi-tagged ALK, 75 nM ULight-Streptavidin (PerkinElmer), 50 nM 6×His-tagged CRBN/DDB1, and 0.5 nM Eu-W1024 Anti-6×His (PerkinElmer) in a buffer consisting of 50 mM Tris, 150 mM NaCl, 1 mM TCEP, 0.02% Tween-20, and 0.5 mg/mL BSA at pH 7.4. The plate was incubated at room temperature for 2 hours, then read on a BMG PHERAstar plate reader with a 337 nm excitation laser and 620 nm and 665 nm emission filters. The TR-FRET signal was calculated as the ratio of emission signals at 665 nm over 620 nm, and the compound-containing wells were normalized to negative controls (0% activity) and 3-(3-{N-methyl[(3-phenyl-1,2,4-oxadiazol-5-yl)methyl]amino}-2,5-dioxo-3-pyrrolin-1-yl)-2,6-piperidinedione (100% activity). Normalized data for each compound were then subjected to a 4-parameter logistic fit. |
| Affinity data for this assay | |
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