Charged nylon membrane

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, and 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 enhanced chemiluminescence (Pierce).

Electrophoresis mobility shift assay probes in a length of 59 nt were commercially synthesized by Tsinke Biological Technology (Hangzhou, China) and labeled with an EMSA Probe Biotin Labeling Kit (Cat No. GS008, Beyotime, Shanghai, China). DNA binding was performed in a 10 μL reaction volume containing EMSA/Gel‐shift binding buffer (Beyotime, Shanghai, China), 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, Shanghai, China), detected by streptovidin‐HRP (Beyotime, Shanghai, China), and finally visualized using the enhanced chemiluminescence (Pierce, Waltham, MA).