Astra version 6

MALS was measured using DAWN HELEOS-II (Wyatt Technology Corporation) downstream of a Shimadzu liquid chromatography system connected to a Superdex 200 10/300 GL (GE Healthcare) gel filtration column. The running buffer for SecB–PhoA complexes was 20 mM KPi (pH 7.0), 100 mM KCl, 4 mM βME, and 0.5 mM EDTA, whereas for SecB–MBP complexes was 20 mM HEPES, pH 7, 150 mM KOAc and 0.05% NaN₃. Protein samples at a concentration of 0.05 to 0.2 mM were used. The flow rate was set to 0.5 ml min⁻¹ with an injection volume of 200 µl and the light scattering signal was collected at room temperature (~23 °C). The data were analyzed with ASTRA version 6.0.5 (Wyatt Technology Corporation).

Protein co-elution by analytical gel filtration alone or coupled to SEC-MALS was carried out using an ÄKTAmicro (GE Healthcare) FPLC at room temperature. Lig PIP mutants incubated with PCNA and DNA were passed over a 3.2/30 Superdex 200 Increase (GE Healthcare) gel filtration column equilibrated in 20 mM MES pH 6.5, 50 mM NaCl, 10 mM MnCl₂ and 0.1 mM TCEP. The 10/300 Superdex 200 Increase gel filtration column was used to analyze complexes undergoing DNA ligation (buffer: 25 mM HEPES pH 7.5, 10 mM MnCl₂, 50 mM NaCl, 1 mM ATP) and complexes of Lig DBD mutants with PCNA (buffer: 25 mM HEPES pH 7.5, 50 mM NaCl, 10 mM MnCl₂, 0.1 mM TCEP).
The Lig-PCNA-DNA molecular mass was measured using the 10/300 Superdex 200 Increase gel filtration column coupled in-line to a UV detector, Dawn HELEOS II MALS flow cell (Wyatt Technology) and OptiLab T-rEX differential refractometer (Wyatt Technology). The buffer contained 20 mM MES pH 6.5, 50 mM NaCl, 10 mM MnCl₂ and the detectors were equilibrated with BSA. Data were processed using ASTRA Version 6.1.6.5 (Wyatt Technology).