GST protein purification and pull down assay

GST tag only and GST-WRKY53 proteins were induced with 200 μM IPTG (Isopropyl β-D-1-thiogalactopyranoside) for three hours at 37°C. HDA9-FLAG protein was purified from HDA9-FLAG transgenic plants using IP methods described above. Purified HDA9-FLAG and GST tagged protein were incubated in the pull down buffer (20 mM Tris-HCl 8.0, 200 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40) for one hour then incubated with Glutathione Sepharose 4B beads (GE Healthcare, 17075601) for one hour at 4°C. After washed with pull down buffer for three times with 5 min per wash, protein-bead complex was boiled in SDS loading buffer, subjected to SDS-PAGE gel, and detected with anti-GST and anti-FLAG antibodies.