Analytical SEC was performed in buffer M (250 mM NaCl, 20 mM HEPES pH 7.0, 1 mM DTT), or in the appropriate NaCl concentration. Samples were mixed at ∼30 µM and incubated for 15 min in 350 mM NaCl, 20 mM HEPES pH 7.0, 1 mM DTT on ice before being filtered and injected onto a GE Superdex 200 10/300-Increase analytical size exclusion column. When appropriate, samples were mixed with 1.1-fold molar excess RNA. All samples were run at 0.35 mL/min, and peaks were analyzed by SDS-PAGE (Invitrogen). For experiments involving reinjection of fractions over SEC, 500 µL of fractions were spin filtered before reinjecting over SEC.
For SEC-MALS, 165 µg of sample was filtered through a 0.1 µm spin filter (Amicon) before being injected onto a preequilibrated KW-804 column (Shodex). For samples with RNA, stoichiometric amounts of RNA were added prior to spin filtration. Data was acquired with an inline DAWN HELEOS MALS and Optilab rEX differential refractive index detector (Wyatt Technology). All analysis was performed using ASTRA VI software (Wyatt Technology). Data was then exported and plotted with R.
For SEC-MALS, 165 µg of sample was filtered through a 0.1 µm spin filter (Amicon) before being injected onto a preequilibrated KW-804 column (Shodex). For samples with RNA, stoichiometric amounts of RNA were added prior to spin filtration. Data was acquired with an inline DAWN HELEOS MALS and Optilab rEX differential refractive index detector (Wyatt Technology). All analysis was performed using ASTRA VI software (Wyatt Technology). Data was then exported and plotted with R.