Minidawn treos mals instrument

SEC-MALS experiments were performed using a fast protein liquid chromatography system (GE Healthcare) connected to a Wyatt MiniDAWN TREOS MALS instrument (Santa Barbara, CA, USA) and a Wyatt Optilab rEX differential refractometer. A Superdex 200 10/300 GL (17-5175-01; GE Healthcare) gel filtration column preequilibrated with 20 mM Tris-HCl (pH 8.0), 100 mM NaCl, and 2 mM DTT was normalized using bovine serum albumin. Individual proteins (HpMsrAB WT, HpMsrA domain, HpMsrB domain, and various mutants, including HpMsrAB^C44S/C318S, HpMsrAB^D197A, HpMsrAB^E193A, HpMsrAB^E339A, and HpMsrAB^Y343F) were prepared separately by the methods described earlier and were injected (1–2 mg/mL, 0.25 mL) at a flow rate of 0.8 mL/min. The final purity of the proteins was checked by SDS-gel electrophoresis (Figure S5). Data were analyzed using the Zimm model for static light scattering data fitting and represented using an EASI graph with a UV peak in the ASTRA V software (Wyatt).

SEC-MALS experiments for MeFDH1 were performed using an FPLC system (GE Healthcare) connected to a Wyatt MiniDAWN TREOS MALS instrument and a Wyatt Optilab rEX differential refractometer. A Superdex 200 10/300 GL (GE Healthcare) gel-filtration column, pre-equilibrated with a final SEC buffer, was normalized using ovalbumin. Proteins (1 mg) were injected at a flow rate of 0.4 ml/min. Data were analyzed using the Zimm model for static light-scattering data fitting and graphed using an EASI graph with a UV peak in the ASTRA V software (Wyatt).

SEC-MALS experiments for Uso1 were performed using an FPLC system (GE Healthcare) connected to a Wyatt MiniDAWN TREOS MALS instrument and a Wyatt Optilab rEX differential refractometer. A Superdex 200 10/300 GL (GE Healthcare) gel-filtration column was pre-equilibrated with buffer A containing 5 mM β-mercaptoethanol and normalized using ovalbumin. Proteins (1 mg) were injected at a flow rate of 0.4 mL/min. Data were analysed using the Zimm model for static light-scattering data fitted and graphed, using EASI graph with a UV peak, in the ASTRA V software (Wyatt).

SEC-MALS experiments were performed using a fast protein liquid-chromatography system (GE Healthcare) connected to a Wyatt MiniDAWN TREOS MALS instrument and a Wyatt Optilab rEX differential refractometer. A Superdex 200 10/300 GL (GE Healthcare) gel-filtration column pre-equilibrated with 20 mM Tris–HCl pH 8.0, 100 mM NaCl, 2 mM DTT was normalized using bovine serum albumin. The individual proteins (streptavidin C1 and C2 and the C-terminal deletion mutants ΔC1 and ΔC2) were prepared separately by the methods described earlier and were injected (1–2 mg ml⁻¹, 0.25 ml) at a flow rate of 0.8 ml min⁻¹. The data were analyzed using the Zimm model for fitting static light-scattering data and were represented using an EASI graph with a UV peak in the ASTRA V software (Wyatt).

SEC-MALS experiments were performed using a fast protein liquid chromatography (FPLC) system (GE Healthcare) connected to a Wyatt MiniDAWN TREOS MALS instrument and an Optilab rEX differential refractometer (Wyatt, Santa Barbara, CA, USA). A Superdex 200 10/300 GL (GE Healthcare) gel-filtration column pre-equilibrated with buffer containing 20 mM HEPES pH 7.5, 150 mM NaCl and 10 mM MgCl₂ was used. FOXL2-DBD, FOXL2–DNA complexes, FOXA1-DBD and FOXO3-DBD proteins were injected (5–7 mg/ml) at a flow rate of 0.4 ml/min. Data were analyzed using the Zimm model for fitting static light-scattering data and graphed using the EASI graph with a UV peak in ASTRA VI (Wyatt).

SEC-MALS experiments were performed using an FPLC system (GE Healthcare) connected to a Wyatt MiniDAWN TREOS MALS instrument and a Wyatt Optilab rEX differential refractometer (Santa Barbara, CA, USA). A Superdex 200 10/300 GL (GE Healthcare) gel-filtration column pre-equilibrated with buffer A was normalized using ovalbumin protein. Proteins were injected (0.5–5 mg) at a flow rate of 0.4 mL/min. Data were analyzed using the Zimm model for fitting static light-scattering data and graphed using EASI graph with a UV peak in ASTRA V software (Wyatt).