2.9 Immunoprecipitation
The crude extracts (with buffer A) of the leaf sheath of the FLAG-CRCT
overexpression line and CRCT knock out line were incubated with
anti-DYKDDDDK tag antibody beads (Fujifilm Wako Chemical) at 4˚C for 1
h. After incubation, the beads were collected using a magnet and washed
three times with a washing buffer (20 mM Tris-HCl pH 7.4, 200 mM NaCl,
2.5 mM MgCl2, 0.05% (w/v) NP-40). FLAG-CRCT associated proteins were
eluted by incubating the beads with 500 μg mL-1DYKDDDDK peptide (Fujifilm Wako Chemical) in 10 mM Tris-HCl pH 7.4, 150
mM NaCl at 4˚C for 30 min. The eluted proteins were then subjected to
SDS-PAGE and identified by LC-MS analysis.