Gfp tag immunomagnetic beads

HEK-293-Flp-In T-Rex cells tagged with EGFP, EGFP-MAB21L1 or mutant EGFP-MAB21L1 were seeded in T-25 flask in culturing media supplemented with 1 μg/ml tetracycline. Cells were harvested by trypsin-EDTA, washed by PBS after 12 hrs of tetracycline treatment and lysed with Nonidet P-40 lysis buffer (50mM Tris, pH 8.0, 150mM NaCl, 1.0% Nonidet P-40) in the presence of protease inhibitor(Roche Applied Science) For each immunoprecipitation, 400 μl of cell lysate were incubated with anti-TBL1XR1 antibody (ab24550,Abcam) and anti-MSI2 antibody(ab76148,Abcam) for 5 h at 4°C. Then 20 μl of Dynabeads protein A (Thermo Fischer) were added and rotated for 2 h at 4°C. Bound immune complexes were washed three times with phosphate-buffered saline. For immunoprecipitation of GFP-tagged proteins, Cell lysis and GFP pulldown was perform using GFP Tag Immunomagnetic Beads (Sino Biologicals) according to manufacturer instructions. The immune-complexes were analysed by Western blotting.

Three biological replicates of HEK-293-Flp-In T-Rex cells tagged with EGFP, EGFP-MAB21L1 or mutant EGFP-MAB21L1 were seeded in T-75 flask in culturing media supplemented with 1 μg/ml tetracycline. Cells were harvested by trypsin-EDTA, washed by PBS after 12 hrs of tetracycline treatment. Cell lysis and GFP pulldown was perform using GFP Tag Immunomagnetic Beads (Sino Biologicals) according to manufacturer instructions. The pull-down beads were subjected to mass spectrometric analysis and raw data was analysed by the MaxQuant and Andromeda software package as described [24 (link)], using the pre-selected conditions for analysis (specific proteases, 2 missed cleavages, 7 amino acids minimum length). Detailed Mass Spectrometry analysis is provided in Supplemental Materials and Methods.