EMSA with NP - Method

EMSA conditions for NP-RNA

Binding buffer: 1x PBS + 0.01% Pluronic + 10% Glycerol

Mix Protein (increasing concentration) + RNA (10-50 nM)

Incubation time: There is an effect of time. Binding equilibrium is not reached after a few minutes. It is recommended to incubate 1h or longer at RT or 37°C.

Gel system: Start with 6-8% PAA/0.4x TBE ; but agarose gels seem to work better - maybe due to the large complexes NP forms.

Protein concentration to use: 10-500nM for full length NP

Binding affinity: of fullength NP will be in the range of 50 nM.

RNA bandshift probes: RNA oligos - Nucleocapsid project

Example picture: XXXXX

Fropm other labs

Described for BCoV

(Lo Wu FEBS J 2019 PMID: 31034708) RNA was titrated with the protein of interest in binding buffer [20 mM HEPES (pH 7.5), 6 mM MgCl2, 1.5 uM EGTA, 22.5 mM NaCl, 330 mM KCl, 36% glycerol, 3.6 mM DTT, 82.5 ug/mL1 BSA] for 15 min at 37 °C with 1 U*mL-1 RNasin (Promega). The bound RNA-protein complexes were separated from unbound RNA by 8% native polyacrylamide gel electrophoresis with crosslink ratio 2.7%, dried, and analyzed by autoradiography.