Anti mbp magnetic beads

MBP- and GST-tagged proteins were both expressed in E. coli strain BL21 (DE3) (plyS). Cells were harvested by centrifugation and resuspended in 20 ml of lysis buffer (50 mM Tris-HCl (pH 8.0) 500 mM NaCl, 0.22 mg/ml lysozyme, 100 μM PMSF, and 10 mM DTT) and sonicated on ice. The lysate was centrifuged, and the supernatant was mixed with Ni-NTA slurry (Qiagen, Venlo, the Netherlands) and rocked for 60 min at 4 °C. The mixture was poured through a Ni-NTA column and washed with 50 ml of phosphate wash buffer (50 mM Tris-HCl (pH 8.0), 20 mM imidazole, 500 mM NaCl, and 10% glycerol). Purified protein was eluted with elution buffer (500 mM imidazole, 1 M NaCl, 10% glycerol, and 50 mM Tris-HCl (pH 8.0)). The imidazole and excess NaCl were removed by dialysis in buffer (50 mM Tris-HCl, 150 mM NaCl and 10% glycerol).
MBP and MBP-EZH2 proteins were incubated at 4 °C with GST-Ku80 proteins overnight. MBP-tagged proteins were captured by incubation with anti-MBP magnetic beads (New England Biolabs, Ipswich, MA, USA) at 4 °C for 3 h. The bead pellet was washed five times with 500 μl of buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% NP-40, 10% glycerol and 2 mM EDTA). Samples were boiled and subjected to SDS-PAGE.

Pull-down assay was performed as described previously (Zhang et al. 2021 (link)). Briefly, the CDS of CsWRKY11 and CsNPR1 were respectively cloned into pGEX-4T-1 and pMAL-c5G plasmids. The reconstructed plasmids were then introduced into Escherichia coli Rosetta (DE3) cells for protein expression. The interacting protein complex was pulled down with anti-MBP Magnetic Beads (NEB), and the pulled-down proteins and input were examined with anti-GST and anti-MBP antibodies.