Gentle ag ab elution buffer

HEK293FT cells were transfected with 3xFLAG-HD-LRRK2 using Lipofectamine 2000 (Life Technologies) for 48 h followed by lysis in buffer containing 20 mM Tris–HCl (pH 7.5), 150 mM NaCl, 3 mM KCl, 10% (v/v) glycerol, 1 mM EDTA, 0.3% (v/v) Triton X-100 supplemented with protease inhibitor cocktail (Roche) and HALT phosphatase inhibitor cocktail (Thermo Scientific). Lysates were spun down at 20 000 g for 10 min, and supernatants were pre-cleared with EZview Red Protein G Affinity Gel for 1 h followed by immunoprecipitation with EZview Red ANTI-FLAG M2 Affinity Gel (both Sigma) for 2 h and gentle washing (six times) with buffer containing 20 mM Tris–HCl (pH 7.5), 150 mM NaCl, 3 mM KCl, and 0.1% Triton. LRRK2 was eluted with Gentle Ag/Ab Elution Buffer (pH 6.6) (Thermo Scientific) with 0.01% (v/v) Triton and 150 μg/ml 3xFLAG^® peptide for 30 min followed by desalting of the eluate with Zeba Spin Desalting Columns (Thermo Scientific) having a 7K molecular mass cutoff. Transfection with siRNA was made using the DharmaFECT1 transfection reagent (GE Healthcare) according to the manufacturer’s instructions. The amount of immunoprecipitated binding partner was normalized to that of the LRRK2 construct eluted from the beads, estimated by densitometry using Image J (https://imagej.nih.gov/ij/).

CHO-K1 cells stably expressing recombinant AER-37 hIgG1 or ligelizumab were grown in glutamine-free medium containing 25 uM msx and 5% dialyzed ultra-low IgG fetal bovine serum in roller bottles for 49 days. Cell culture medium was collected every seven days and replaced with fresh medium. Conditioned medium was concentrated and dialyzed into 50 mM Tris, 150 mM NaCl, pH 7.0. This material was adsorbed to GammaBind-Plus Sepharose (GE Life Sciences); IgG was was eluted with Gentle Ag/Ab Elution Buffer (ThermoFisher Scientific), dialyzed into 150 mM NaCl, and concentrated using an Amicon stirred cell.