Assay Method Information

Assay Name:  TR-FRET cereblon binding assay
Description:  The 6XHis-tagged full length human CRBN bound to full length human DDB1 used in the assay was purified as described elsewhere with the exception that the thrombin cleavage/ortho nickle step was removed.8b In the assay, 60 nM 6Xhis-tagged CRBN-DDB1 was combined with 30 nM cy5-conjugated cereblon modulator (compound 7) and 3 nM LanthaScreen Eu-anti-His Tag antibody (ThermoFisher catalogue no. PV5596) in 20 mM HEPES pH 7, 150 mM NaCl, 0.005% Tween-20 assay buffer. FRET was observed by exciting at 340 nm and monitoring emission at 615 nm (non-FRET emission) and 665 nm (FRET emission), and FRET efficiency was determined by the ratio of FRET to non-FRET emission (665 nm/615 nm).
Affinity data for this assay

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