Akta pure

The oligomeric state of tag-removed, fully glycosylated SPACA6 was assessed by SEC-MALS. 0.14 mg Bovine Serum Albumin (BSA) and 0.14 mg SPACA6 were prepared in 1X PBS at a concentration of 1.2 mg mL⁻¹. A Superdex 75 10/300 GL size-exclusion column (Cytiva/GE) was equilibrated overnight with 5 CV PBS. Monomeric BSA (MW = 66,432 Da) was used as a reference calibration standard. Prior to SEC-MALS analysis, each sample was centrifuged at 15,000 × g for 15 min at 4 °C and then the supernatant was loaded onto the size-exclusion column on an AKTA Pure FPLC (Cytiva) at 0.2 mL min⁻¹. Triple detection was performed by measuring absorbance at 280 nm using the integrated UV monitor on the AKTA Pure, three-angle light scattering using the miniDAWN TREOS MALS detector (Wyatt) and refractive index (RI) using Optilab T-rEX RI detector (Wyatt). The data were processed, and weight-averaged molecular mass was calculated using the ASTRA software package (version 7.0.2.11).

The HHARI/UbcH7 complex was prepared at 1.5mg/ml in 25 mM MOPS, 150 mM NaCl at pH 7.5 and placed in an auto sampler a 4°C. 25 μl sample volumes were injected onto a GE Superose 6 Increase 3.2/300 mm column (2.4ml) equilibrated with matched buffer. Experiments were conducted at room temperature (∼25°C) with a flow rate of 0.1ml/min. Absorbance at 280 nm, light scattering, and change in refractive index were measured with a GE AKTA Pure coupled to a Wyatt miniDAWN TREOS and Optilab T-rEX differential refractive index detector. Molar mass was calculated from the Raleigh ratio based on multi-angle (static) light scattering and protein concentration from the change in refractive index (dn/dc = 0.185). Analysis was performed using Wyatt ASTRA VI software. Finally, absorbance at 280 nm and molar mass distribution were plotted as a function of elution volume (ml) using Excel (Microsoft).