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.