TWIN-Strep tagged proteins were then purified by incubating whole cell lysates from the transiently-transfected cell lines with 50 μl of MagStrep “type3” XT beads (IBA Life Sciences) for two hours at 4 °C. Magnetic resin was washed three times in 20 mM HEPES pH 7.9, 2 mM MgCl2, 0.2 mM EGTA, 10% glycerol, 0.1% NP-40, 0.2 M NaCl, 0.1 mM PMSF, and 1 mM DTT. Proteins were eluted with 1X Buffer BX (IBA LifeSciences) which contains 10 mM D-biotin. Purified proteins were visualized on a NuPAGE Bis-Tris polyacrylamide gel (ThermoFisher) and then transferred to Immobilon-FL Hydrophobic PVDF Transfer Membrane (Millipore Sigma) with subsequent immunoblotting against either the FLAG tag or TWIN-Strep tag (Anti-FLAG M2, Sigma-Aldrich; THETM NWSHPQFEK antibody, GenScript). For immunoblots against endogenous DALRD3, Proteintech’s DALRD3 antibody was used.
