2.6 Pull-Down Experiments
For the pull-down assay, glutathione S-transferase (GST)-CsHSP24.6 and free GST proteins were coupled to GST Bind Resin (NEB, E8021L). Maltose-binding protein (MBP)-CspTAC5 and free MBP proteins were incubated with GST-CsHSP24.6 and free GST coupled to GST Bind Resin in a pull-down buffer (50 mM Tris–HCl and 100 mM NaCl) at 4 °C for 3 h. After incubation, the beads were washed five times with ice-cold pull-down buffer, and the bound proteins were eluted in pull-down buffer containing an additional 20 mM L-glutathione (reduced) and 2.5 mM CaCl2. The eluted proteins were resolved by SDS–PAGE and analyzed by immunoblot43.