Nylon filter

The filter-binding assay was performed as described previously (Rio 2012 (link)). Of note, 62.4 pmol of purified RNA was 5′-end labeled with 5 µCi [γ-³²P] ATP by T4 polynucleotide kinase (New England Biolabs) in nuclease-free buffer at 37°C for 1 h. The reaction was stopped by incubating at 70°C for 10 min. Labelled RNA was purified by mini Quick Spin Oligo Columns (Roche). The purified oligonucleotides were then heated at 95°C for 10 min and cooled down for 30 min. Different concentrations of protein were titrated to a constant amount of 50 nM CAG72 RNA in a final volume of 50 µL and incubated at room temperature for 1 h in binding buffer consisting of 10 mM Tris 7.5, 50 mM KCl, 750 µM MgCl₂, 100 µM EDTA, 5% glycerol, 600 µM dithiothreitol, 0.1 mg/mL tRNA, and 40 µg/mL BSA. Nitrocellulose (0.45 µm pore size, Bio-Rad) and nylon filter (Roche) were presoaked in wash buffer 10 mM Tris 7.5, 50 mM KCl, and 1 mM dithiothreitol for 2 h. Both filters, Nitrocellulose on top and nylon filter at the bottom, were placed into a dot-blot apparatus (Bio-Rad). The wells were washed once and vacuum was applied before the samples were loaded. The wells were washed eight times with 100 µL washing buffer after samples were loaded. The Nitrocellulose filters were analyzed using auto-radiography to measure the retained radiolabeled RNA on the Nitrocellulose filter.