Anti dykddddk mouse mab

In vitro biochemical interaction assay was performed as described previously^{10 (link)}. 10 µL CRBN mixtures containing 0.5 µL biotinylated bls-CRBN in AlphaScreen buffer (100 mM Tris [pH 8.0], 0.01% Tween20, 100 mM NaCl, and 1 mg/mL bovine serum albumin [BSA]) were prepared. 5 µL compound mixtures containing thalidomide derivatives were prepared in AlphaScreen buffer. 5 µL substrate mixtures containing 0.8 µL FLAG-GST-SALL4, -IKZF1 or -PLZF in AlphaScreen buffer were prepared. Then, the three mixtures were dispensed and incubated at 26 °C for 1 h in a 384-well AlphaPlate (PerkinElmer). Subsequently, a 5 µL detection mixture containing 0.2 µg/mL anti-DYKDDDDK mouse mAb (#012-22384, FUJIFILM Wako Pure Chemical Corporation), 0.08 µL streptavidin-coated donor beads (#6760617, PerkinElmer, MA, USA), and 0.08 µL Protein A-coated acceptor beads (#6760617, PerkinElmer) in AlphaScreen buffer were added to each well and incubated. After incubation at 26 °C for 1 h, luminescence signals were detected using an EnVision plate reader (PerkinElmer version 1.12).

IMiD at a final concentration of 50 µM and 0.5 µl biotinylated CRBN were mixed in 15 µl AlphaScreen buffer containing 100 mM Tris (pH 8.0), 0.01% Tween20, 100 mM NaCl, and 1 mg/ml BSA. Then, 5 µl of a mixture containing 0.8 µl FLAG-GST-Venus-m1, -m2, or -m3 in AlphaScreen buffer was added, and the reaction mixtures were incubated at 26 °C for 1 h in a 384-well AlphaPlate (PerkinElmer). Subsequently, 5 µl of a detection mixture containing 0.2 µg/ml anti-DYKDDDDK mouse mAb (Wako), 0.08 µl streptavidin-coated donor beads, and 0.08 µl Protein A-coated acceptor beads (PerkinElmer) in AlphaScreen buffer was added to each well. After incubation at 26 °C for 1 h, luminescence was read on an EnVision plate reader (PerkinElmer).