| Assay Method Information | |
| | Time-Resolved Fluorescence Resonance Energy Transfer CRBN Competitive Binding Assay |
| Description: | The supernatant was collected and clarified as above. For protein expression, 5 mL P2 virus was added to each liter of Sf cells (2×106 cells/mL) and shaken at 27° C. for three days. Cells containing expressed protein were pelleted by centrifugation at 500×g for 5 min, the supernatant was removed, and pellets were stored at −80° C. until processing. Cells were resuspended in lysis buffer [50 mM Tris (pH 7.5), 500 mM NaCl, 1 mM TCEP, 10 mM imidazole, 10% glycerol] containing SIGMAFAST Protease Inhibitor Cocktail Tablets, EDTA-Free (Sigma-Aldrich) and 125 U/mL Benzonase Nuclease (Sigma-Aldrich). The suspension was incubated on ice for 1 h, gently sonicated, and centrifuged for 1 h at 40,000×g at 4° C. The supernatant was applied to Ni-NTA Superflow resin (QIAGEN) equilibrated with lysis buffer. The resin was washed with 10 column volumes (CV) of lysis buffer, and proteins were eluted with 10 CV lysis buffer containing 500 mM imidazole. The solution was diluted to 200 mM NaCl using lysis buffer without NaCl and applied to a HiTrap ANX FF (High Sub) column (Cytiva) equilibrated with ANX binding buffer [50 mM Tris (pH 7.5), 200 mM NaCl, 3 mM TCEP]. The column was washed with 10 CV ANX binding buffer followed by 10 CV ANX wash buffer [50 mM Bis-Tris (pH 6.0), 200 mM NaCl, 3 mM TCEP]. A 20 CV linear gradient was then immediately applied from ANX binding buffer to ANX binding buffer containing 1 M NaCl using an AKTA avant chromatography system (Cytiva). The purest fractions were applied to a HiLoad 26/600 Superdex 200 pg size exclusion column equilibrated with 10 mM HEPES (pH 7.0), 240 mM NaCl, and 3 mM TCEP. |
| Affinity data for this assay | |
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