Accugel 29 1

Complexes between U2 and U6 RNAs and the Lsm2-8 protein were formed in a volume of 10 μL by mixing U2 and U6 oligos in RNA dilution buffer (100 mM KCl, 20% w/v sucrose, 20 mM HEPES (pH 7), 1 mM EDTA (pH 8), 1 mM TCEP·HCl, 0.01% v/v Triton X-100, 0.2 mg/mL yeast tRNA [Ambion, Catalog No. AM7119], 0.2 mg/mL sodium heparin [Sigma Aldrich, Catalog No. H4784]) together with purified Lsm2-8 in protein dilution buffer (100 mM KCl, 20% w/v sucrose, 20 mM HEPES (pH 7), 1 mM EDTA (pH 8), 1 mM TCEP·HCl, 0.01% v/v Triton X-100, and 0.2 mg/mL BSA [Pierce, Catalog No. 23209]) in equal volume. Final concentrations for U6, U2 oligos, and purified Lsm2-8 are described in the corresponding figures. All reactions were incubated at room temperature (22–24°C) for 30 min prior to gel electrophoresis.
After 30 min, the reactions were placed on ice and then loaded onto a native 6% polyacrylamide gel (AccuGel 29:1, National Diagnostics), which had been pre-run with 1× TBE buffer at 300 V for 30 min at 4°C. Electrophoresis was then carried out at 300 V for 2–4 h. For detection of radioactive bands, the gels were dried and then exposed to a phosphor screen. Phosphor images of the screens were taken in the phosphorescence mode of a Typhoon scanner (Cytiva). Results were analyzed with ImageQuantTL (Cytiva) software.