Electrophoresis mobility shift assay (EMSA)
The flag-OsNF-YB1 protein was expressed using Gzl Custom Cell Free
Protein Expression Kit (GZL bioscience). DNA probes in a length of 59 nt
were commercially synthesized by GENEWIZ Biological Technology and
labeled with an EMSA Probe Biotin Labeling Kit (Beyotime, Cat No.
GS008,). DNA binding was performed in a 10 L reaction volume containing
EMSA/Gel-shift binding buffer (Beyotime), 2 nmol biotin-labeled probe, 5
nmol purified recombinant protein. Non-labeled DNA oligos were used as
competitor. Recombinant protein was pre-incubated with the EMSA/Gel-
shift binding buffer for 20 min at 25 °C prior to the addition of the
biotin-labeled probe and further incubated at 25 °C for 20 min. A 6%
(W/V) polyacrylamide gel was pre-run for 30 min, and then the binding
reaction is subjected to gel electrophoresis. The DNA probes were then
transferred to a charged nylon membrane (Beyotime), detected by
streptovidin-HRP (Beyotime), and finally visualized using the enhanced
chemiluminescence (Pierce).