DmPlk4 PB3 was purified without cleaving the N-terminal His6 tag, concentrated to either 10 or 27 mg/ml, and exchanged into running buffer (25 mM HEPES, pH 7.5, 300 mM NaCl, 0.1% β-ME, and 0.2 g/l NaN3). A Superdex 200 10/300 GL gel filtration column (Cytiva) was equilibrated in the running buffer, and 100 µl samples of protein were injected onto the column. Eluate was passed in tandem through a Wyatt DAWN HELEOS II light-scattering instrument and a Wyatt Optilab rEX refractometer (Wyatt, 1993 ). The light-scattering and refractive index data were used to calculate the weight-averaged molar mass of each peak using the Wyatt Astra V software program (Wyatt Technology). Two DmPlk4 PB3 and HsPlk4 PB3 runs were performed for each purification scheme, with two purifications for each construct, for a total of eight experiments.
Wyatt astra 5
Manufactured by Wyatt Technology
The Wyatt Astra V is a multi-angle light scattering (MALS) detector designed for the characterization of macromolecules and nanoparticles in solution. It measures the intensity of scattered light at multiple angles to determine the molar mass, size, and conformation of analytes.
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3 protocols using wyatt astra 5
1
Characterizing DmPlk4 PB3 and HsPlk4 PB3 by SEC-MALS
2
Size Exclusion Chromatography and Light Scattering
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Full-length and truncated SPR2 constructs were individually injected onto a Superdex 200 10/300 GL size exclusion column (GE Healthcare) preequilibrated and passed through a storage buffer supplemented with 0.2 g/L sodium azide. Samples were subsequently passed consecutively through a Wyatt DAWN HELEOS II light scattering instrument and a Wyatt Optilab rEX refractometer. The light scattering and refractive index values were used to calculate the weight-averaged molar mass (MW) and the mass fraction in each peak using the Wyatt Astra V software program (Wyatt Technology Corp.).
3
Molar Mass Analysis of Plk4 Proteins
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DmPlk4 PB3 was purified without cleaving the N-terminal His6 tag, concentrated to either 10 or 27 mg/mL, and exchanged into running buffer (25 mM HEPES, pH 7.5, 300 mM NaCl, 0.1% β-ME, and 0.2 g/L NaN3). A Superdex 200 10/300 GL gel filtration column (Cytiva) was equilibrated in the running buffer, and 100µL samples protein were injected onto the column.
Eluate was passed in tandem through a Wyatt DAWN HELEOS II light scattering instrument and a Wyatt Optilab rEX refractometer (Wyatt, 1993) . The light scattering and refractive index data were used to calculate the weight-averaged molar mass of each peak using the Wyatt Astra V software program (Wyatt Technology) . Two DmPlk4 PB3 and HsPlk4 PB3 runs were performed for each purification scheme, with two purifications for each construct, for a total of eight experiments.
Eluate was passed in tandem through a Wyatt DAWN HELEOS II light scattering instrument and a Wyatt Optilab rEX refractometer (Wyatt, 1993) . The light scattering and refractive index data were used to calculate the weight-averaged molar mass of each peak using the Wyatt Astra V software program (Wyatt Technology) . Two DmPlk4 PB3 and HsPlk4 PB3 runs were performed for each purification scheme, with two purifications for each construct, for a total of eight experiments.
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