Minidawn treos malls detector

FLAG-T. castaneum methoprene-tolerant and His₆-TcTAI were coexpressed and purified as described previously. Purified proteins at ∼5 mg/ml were subjected to size-exclusion chromatography using a Superdex 200 10/300 column (GE Healthcare) at a flow rate of 0.5 ml/min in 50 mM Tris, 350 mM NaCl, 10 μM methoprene, 2 mM MgCl₂, 100 μM TCEP, pH 8.0, with UV monitoring at 280, 254, and 215 nm. Fractions were collected in aliquots of 0.5 ml. Light scattering and concentration data were collected by in-line miniDawn TREOS MALLS detector and Optilab T-rEX refractometer (Wyatt Technology), respectively. The weight-average molecular weights were calculated using the intensity of the scattered light in combination with the change in refractive index, whereas the protein concentration at the detector was determined by the change in refractive index. UV, MALLS, and differential refractive index data were collected using the ASTRA software (Wyatt Technology). Molecular weight determinations were also done with the ASTRA software according to the Zimmerman model using the standard dn/dc value of 0.1852 ml/g for proteins (85 ).

For MALLS, purified hStau1 and truncations were loaded onto a Superdex 200 Increase 10/300 GL column (GE Healthcare), and connected to a miniDAWN TREOS MALLS detector and Optilab T-rEX differential refractometer (Wyatt Technologies). Runs were performed in buffer (20 mM Tris–HCl, pH 7.5, and 150 mM KCl, at RT). For the proteins alone, 200 μg (FL), 400 μg (hStau1^1–360), 430 μg (hStau1^182–360), or 350 μg (hStau1^182–577) protein were injected on the column. For RNA–protein complexes, 140 μg of wt or truncated protein were pre-incubated with a 1.2 M excess of ARF1 SBS dsRNA. Molecular weight calculations were performed using ASTRA software (Wyatt Technologies).