Heleos8 multi angle light scattering detector

Proteins were mixed at the indicated ratios and equilibrated in 50 mM HEPES pH 7.5, 100 mM NaCl, 1 mM MgCl₂, 1 mM Dithiothreitol (DTT) and 10% glycerol (v/v) buffer supplemented with 1 mM adenosine diphosphate (ADP), ATP or AMPPNP for 3 h at room temperature. A total of 100 μl of these mixtures (containing 50–150 μg of total protein) were loaded onto either a Superose 6 HR10/30 column, or a Superdex 200 HR10/30 column (GE), equilibrated with the same buffer lacking glycerol and nucleotides. The separation was conducted at a flow rate of 0.5 ml/min. Presence of DTT or Tris(2-carboxyethyl)phosphine (TCEP) as reductants did not influence the results. Size exclusion chromatography and multi-angle light scattering (SEC-MALS) analysis was performed at 22°C using a Shimadzu (Kyoto, Japan) chromatography system, connected in-line to a Heleos8+ multi angle light scattering detector and an Optilab T-rEX refractive index (RI) detector (Wyatt Technologies, Goleta, CA, USA). Results were processed and analysed using ASTRA 6 (Wyatt Technologies).

Purified proteins were fractionated on a Superose 6 10/300 GL or a Superose 12 10/300 column equilibrated with 50 mM HEPES, pH 7.5 buffer containing 100 mM NaCl, 1 mMDTT, 1 mM EDTA, at flow rate of 0.5 ml/min. 500 µl samples containing analysed proteins were injected on the column and run at a flow rate of 0.5 ml/min. SEC-MALS analysis was performed at 20°C using a Shimadzu (Kyoto, Japan) chromatography system, connected in-line to a Heleos8+ multi angle light scattering detector and an Optilab T-rEX refractive index (RI) detector (Wyatt Technologies, Goleta, CA). Protein samples in 50 mM HEPES pH 7.5, 100 mM NaCl, 1 mM DTT, 1 mM EDTA, 10% glycerol, were injected in this system, and the resulting MALS, RI and UV traces processed in ASTRA 6 (Wyatt Technologies).