To verify the binding activity of the RdRp complex, an electrophoretic mobility shift assay was performed. The RdRp complex was preincubated at 4°C overnight and then incubated with S1*/S2 RNA primer/template in EMSA binding buffer (Thermo Fisher Scientific, Waltham, MA, USA) for 30 min at 30°C. Samples were resolved on 6% native polyacrylamide gels running in 0.5×TBE buffer at 120 V for 45 min in a 4°C room and then transferred to nylon membrane (Amersham Biosciences) running in 0.5×TBE buffer at 15 V for 40 min. The biotin-labeled RNA was detected by LightShift™ EMSA Optimization& Control Kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s protocol and imaged by the ChemiDoc™ Imaging System (Bio-Rad Laboratories).
Emsa binding buffer
Manufactured by Thermo Fisher Scientific
Sourced in United States
EMSA-binding buffer is a laboratory reagent used in Electrophoretic Mobility Shift Assay (EMSA) experiments. It is designed to maintain the stability and integrity of protein-DNA complexes during the electrophoresis process.
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3 protocols using emsa binding buffer
1
Electrophoretic Mobility Shift Assay for RdRp Binding
2
Purification and Characterization of BES1-MBP
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The amplified coding sequences of BES1 were fused in-frame with MBP tags and transformed into Escherichia coli. BES1-MBP recombinant proteins were purified. MBP was purified as the control. Recombinant proteins were then incubated with biotin-labeled probes, or with corresponding unlabeled probes for 30 min in EMSA-binding buffer (Thermo Fisher Scientific). Reaction mixtures were separated by non-denaturing polyacrylamide. DNA signals were detected by chemiluminescence.
3
Purification and Characterization of BES1-MBP
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The amplified BES1 coding sequence was cloned in frame with the MBP gene and introduced into Escherichia coli. The production of the recombinant proteins BES1-MBP and MBP was induced by 0.4 mM isopropyl-β-D-thiogalactopyranoside (IPTG) for 24 h at 16°C. Purified recombinant proteins were incubated with biotin-labeled probes or with corresponding unlabeled probes for 30 min in EMSA-binding buffer (Thermo Fisher). Reaction mixtures were separated on non-denaturing polyacrylamide gels, and DNA signals were detected by chemiluminescence. Probes used in this assay are listed in Supplemental Table S1 .
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