Cy3 labeled RNA was renatured in 1× folding buffer for 5 min at 95°C and slowly cooled (0.1°C/sec) to 4°C. RNA was mixed with increasing concentration of protein (0–5000 nM) and incubated for 15 min at room temperature. The final concentration of RNA was 0.5 nM. Samples were loaded on 8%–10% polyacrylamide gel (19:1 acrylamide/bisacrylamide ratio), and electrophoresis was carried out in 0.5× TB at 4°C (DNAPointer, BioVectis) followed by imaging with an Amersham Typhoon laser-scanner (Cytiva). Averaged data from at least three independent experiments were fitted to the Hill equation using Origin (OriginLab) software.
For qualitative EMSA, 25 pmol of RNA was renatured in 1× folding buffer for 5 min at 95°C and slowly cooled (0.1°C/sec) to 4°C. RNA was mixed with increasing concentration of protein (10–100 pmol) and incubated for 25 min at room temperature. The final concentration of RNA was 1.25 µM. Samples were loaded on 8%–10% polyacrylamide gel (19:1 acrylamide/bisacrylamide ratio) and electrophoresis was carried out in 0.5× TB at 4°C (DNAPointer, BioVectis). RNA was visualized with Toluidine Blue or SYBR Gold staining.
For qualitative EMSA, 25 pmol of RNA was renatured in 1× folding buffer for 5 min at 95°C and slowly cooled (0.1°C/sec) to 4°C. RNA was mixed with increasing concentration of protein (10–100 pmol) and incubated for 25 min at room temperature. The final concentration of RNA was 1.25 µM. Samples were loaded on 8%–10% polyacrylamide gel (19:1 acrylamide/bisacrylamide ratio) and electrophoresis was carried out in 0.5× TB at 4°C (DNAPointer, BioVectis). RNA was visualized with Toluidine Blue or SYBR Gold staining.