Astra 5 software vs 5

Analysis of the aggregation status of labeled and not labeled γ-C and KTI (2 mg/mL) was performed by using the serial system composed by a HPLC pump (515 HPLC pump, Waters), a SEC column (Superose 12, 10/300, GE Healthcare, Milan, Italy), a UV spectrophotometer (Dual λ absorbance detector 2487, Waters), a multi-angle light scattering device (DAWN HELEOS, Wyatt, Santa Barbara, CA, USA), provided with a fast photon counter (QELS), and a differential refractometer (Waters), as previously described in Caparo et al. [21 (link)]. Two hundred µL of samples, previously centrifuged to remove the insoluble fraction, were injected with PBS (pH 7.5) as mobile phase, at a flow rate of 0.5 mL/min. Molar masses were calculated by means of the Astra V software vs. 5.3.4.20 (Wyatt), using a dn/dc value of 0.185 for γ-C samples.

Size-Exclusion Chromatography (SEC) combined with Multi-Angle Light Scattering (MALS) detection was performed in an HPLC system composed by a Waters 515 HPLC Pump, a Waters 2487 Dual λ Absorbance detector (Waters, Sesto San Giovanni, Italy), a Wyatt Dawn Heleos MALS and a Wyatt Optila T-rEX differential refractive index detector (Wyatt Technology, Santa Barbara, CA, USA).
200 μL of 2 mg/mL GCAP1 samples were run on a Superdex 75 Increase 10/300 GL column (GE Healthcare, Milan, Italy), by using 20 mM Tris-HCl, pH 8, 5 mM 2-mercaptoethanol, with/without 1 mM CaCl₂, as mobile phase, at a flow rate of 0.8 mL/min. Molar masses were calculated by means of the Astra V software vs. 5.3.4.20 (Wyatt), using a dn/dc value of 0.185.