Wyatt optilab rex differential refractometer

Size-exclusion chromatography coupled to multi-angle light scattering (SEC-MALS) experiments were performed by injecting 100 μL samples onto an Agilent Bio SEC-5 500 Å column. Light scattering was measured in-line using a Wyatt HELEOS-II 18-angle photometer coupled to a Wyatt Optilab rEX differential refractometer (Wyatt Technology Corp). Ste2-mini Gpa1 heterotrimer complex, Ste2-wild type Gpa1 heterotrimer complex, Ste2, and mini-Gpa1 heterotrimer were injected at 0.36 mg/mL, 0.2 mg/mL, 0.18 mg/mL and 0.47 mg/mL, respectively. Analytical SEC of Ste2-mini Gpa1 complex, Ste2-wild type Gpa1 complex, and Ste2 were performed at 25°C in a buffer containing 20 mM HEPES pH 7.5, 100 mM NaCl, 10 mM MgCl₂, 1 μM α-factor, 0.001% (w/v) LMNG, 0.0005% (w/v) CHS. Mini-Gpa1 heterotrimer was characterised in 20 mM HEPES pH 7.5, 100 mM NaCl, 10 mM MgCl₂, 0.01 mM GDP. BSA was used for calibration. Molecular masses were determined using the three-detector methodology⁵⁸ implemented in the ASTRA software package (Wyatt Technology). Extinction coefficients of 1.218, 1.154, 1.157 and 1.129 for Ste2-mini G protein heterotrimer, Ste2-wild type G protein heterotrimer, Ste2, and mini-Gpa1 heterotrimer, respectively, were calculated in PROTPARAM. A dn/dc value of 0.139 was used for the detergent micelle⁵⁹ . The data was plotted in GraphPad Prism 8.

AP-1 core was incubated with Arf1 and tetherin-Nef at 4 °C overnight in 20 mM Tris, pH 8.0, 200 mM NaCl, 0.3 mM TCEP, 5 mM MgCl₂, and 1 mM GTP. The molar ratio of AP-1 core: Arf1 (17-181) Q71L: tetherin-Nef was fixed at 1:4:6. The final concentration of AP-1 core was 4 mg/ml (20 μM). SEC-MALS experiments were performed using an Agilent 1200 HPLC system (Agilent Technologies, Santa Clara, CA), coupled to a Wyatt DAWN HELEOS-II MALS instrument and a Wyatt Optilab rEX differential refractometer (Wyatt, Santa Barbara, CA). For chromatographic separation, a WTC-050S5 size-exclusion column (Wyatt) with a 20 μl sample loop was used at a flow rate of 0.3 ml/min in the buffer of 1x PBS, pH 7.4, 5 mM MgCl₂, 0.2 mM TCEP. The outputs were analyzed by the ASTRA V software (Wyatt). MALS signals, combined with the protein concentration determined by refractive index, were used to calculate the molecular mass of AP-1:Arf1:tetherin-Nef complex and AP-1 alone.