Electrophoretic mobility shift assay (EMSA)
Cy5-labelled double strand DNA fragments containing CATGTG motif and its
mutated version (GGGGGG) were artificially synthesized as probes in
EMSA. CaNAC2c-GST or GST proteins were incubated with wild-type or
mutated probe which was labelled with Cy5 fluorochrome in 5×binding
buffer (1 M Tris-HCl pH 7.5, 5 M NaCl, 1 M KCl, 1 M
MgCl2, 0.5 M EDTA pH 8.0, 10 mg ml-1BSA). The mixture was separated by PAGE gal and then scanned by
Odyssey@ CLX (LI-COR, USA).