Optilab t rex system

Size exclusion chromatography (SEC) combined with multi-angle light scattering (MALS) and refractometry (RI) experiments were performed with a Shodex KW405–4F size exclusion column equilibrated with 50 mM Tris pH 8.0, 200 mM NaCl, 1 mM DTT and 5% (v/v) glycerol. 12.5 μl of protein samples of HpDnaB or mutants (15 mg.ml⁻¹) and HpDnaG^HBD (20 mg.ml⁻¹) were injected onto the column. For samples containing nucleotides the proteins were first incubated with 5 mM ATP or AMPPNP and 5 mM MgCl₂ and the buffer was supplemented with 0.5 mM of the corresponding nucleotides and 5 mM MgCl₂. For samples of the HpDnaB•HpDnaG^HBD complex, the separated proteins were incubated at equal molar amounts at a final concentration of 15 mg.ml⁻¹. For ssDNA complex measurements the protein or complexes were first mixed with a 20mer polydT oligonucleotide at either 45 μM or 75 μM (final concentration). On-line MALS detection was performed with a miniDAWN-TREOS detector (Wyatt Technology Corp., Santa Barbara, CA) using a laser emitting at 690 nm and refractive index measurements were performed using an Optilab T-rex system (Wyatt Technology Corp., Santa Barbara, CA). Weight averaged molar masses were calculated using the ASTRA software (Wyatt Technology Corp., Santa Barbara, CA).