Minidawn treos three angle light scattering detector

MALS experiments were performed at 18°C. Samples were passed over a Superdex 200 increase 10/300 (His₆-MORF1^79–190) or a Superdex 75 10/300 gel filtration column (MORF1^79–190) coupled to a miniDAWN TREOS three-angle light scattering detector (Wyatt Technology) and a RefractoMax520 refractive index detector (ERC). Detectors were aligned, corrected for band broadening, and photodiodes were normalized with BSA as a reference. For calculation of the molecular mass, protein concentrations were determined from the differential refractive index with a specific refractive index increment (dn/dc) of 0.185 ml/g. Data were analyzed with ASTRA 6.1.4.25 (Wyatt Technology). For analytical gel filtration, MORF1^79–190 or MORF1^86–186 were passed over a Superdex 75 10/300 gel filtration column and UV absorbance at 280 nm was recorded.

Purified IMP3 RRM12 (in buffer 20 mM Tris pH 7.5, 150 mM NaCl, 1 mM DTT) was concentrated to 5 mg ml⁻¹ and loaded to a Superdex 200 10/300 GL gel-filtration column (GE Healthcare) equilibrated in 20 mM Tris pH 7.5, 200 mM NaCl, 0.02% w/v NaN₃, and 1 mM DTT. Light scattering was recorded with an in-line miniDAWN TREOS three angle light scattering detector (Wyatt Technology), and protein concentration was detected using an in-line Optilab T-rEX refractive index detector (Wyatt Technology). The molecular mass of IMP3 RRM12 was calculated using ASTRA 6 software (Wyatt Technology).