Superdex 75 increase 10 300 gl column

Size exclusion chromatography was performed using a Superdex 75 Increase 10/300 GL column (Cytiva) equilibrated in phosphate‐buffered saline (PBS, pH 7.4). A total volume of 500 μL of the protein sample was injected into the column. The chromatographic separation was carried out with PBS as the running buffer, and the column was run over 1.5 column volumes to ensure adequate separation. Protein elution was monitored continuously by measuring the absorbance at 280 nm.

The purified samples were characterized by fast protein liquid chromatography SEC. Equimolar amounts (5 µM) of samples prepared in PBS (pH 7.4) were injected to Superdex 75 Increase 10/300 GL column (Cytiva, Uppsala, Sweden) and eluted at a flow rate of 0.2 mL/min. Ovalbumin, conalbumin, and aldolase from Gel Filtration Calibration Kit HMW (Cytiva, Uppsala, Sweden) were used as standard proteins. The analysis was performed using of NGC Quest 10 Chromatography System (Bio-Rad, Hercules, CA, USA).

To determine the oligomeric state of KaiB^RS-3m and KaiB^TV-4, 100 μl of 500 μM purified protein was loaded onto a Superdex 75 increase 10/300 GL column (Cytiva) equilibrated at 0.25 ml min⁻¹ flow rate (AKTA HPLC system) (Extended Data Fig. 4) in 100 mM MOPS, pH 6.5, 50 mM NaCl, 2 mM TCEP. Detection was performed using a MiniDAWN multi-angle light-scattering detector and an Optilab differential refractometer (Wyatt Technology). Molecular masses were calculated using Astra (v.8.1.2.1) using a differential index of refraction (dn/dc) value of 0.185 ml g⁻¹.

For SEC-MALS analysis, the GST-tag was removed from Afadin-CC by PreScission (3C) protease cleavage, using 30 µg GST-3C per 1 mg GST-Afadin-CC in 50 mM Tris, pH 7.4, 150 mM NaCl, 5% (v/v) glycerol, 5 mM DTT, 0.5 mM EDTA. Cleavage reactions were performed for 16 hr at 4°C, followed by removal of GST-3C and cleaved GST by reabsorption onto glutathione sepharose beads for 1 hr at 4°C. Cleavage reactions were then passed through a gravity column to separate sepharose beads from eluate containing the cleaved Afadin-CC. Afadin-CC was immediately loaded onto a HiLoad Superdex 75 pg 16/600 column (Cytiva) equilibrated in SEC buffer (50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 5% [v/v] glycerol, 5 mM DTT) and peak fractions concentrated in 3K MWCO centrifugal filter unit (Merck Millipore) to 1 mg/ml (120 µM).
A 100 µl sample at 1 mg/ml was injected onto a Superdex 75 Increase 10/300 GL column (Cytiva) equilibrated in SEC buffer at a flow rate of 0.5 ml/min. Data for static light scattering and differential refractive index were measured in-line using DAWN 8+ and Optilab T-rEX detectors, respectively (both Wyatt Technology). The absolute molar masses of the elution peaks were calculated in ASTRA 6 (Wyatt Technology) using a protein dn/dc value of 0.185 ml/g.

SEC was performed with a Superdex 75 Increase 10/300 GL column, 10 × 300 mm (Cytiva, Marlborough, MA, USA), where the mobile phase was 50 mM pH 7.0 phosphate buffer containing 400 mM sodium chloride at a flow rate of 0.8 mL/min and a column temperature of 30 °C. Samples were prepared as triplicates by dissolving the protein powder, physical mixture, or protein-loaded extrudates in the mobile phase with a target concentration of 3 mg/mL protein. Human insulin containing samples were pre-dissolved in 0.05 M hydrochloric acid aqueous solution and then diluted with the mobile phase. The UV detector (SPD-40 UV Detector, Shimadzu, Japan) wavelength was 214 nm. Sample vials were cooled at 15 °C in the autosampler. 20 μL of each sample was injected and potential aggregate and/or fragment formation after processing was analyzed in comparison with a chromatogram of the freshly prepared protein powder and physical mixture samples.

Protein sequences for Pfs25 (PF3D7_1031000) and Pfs28 (PF3D7_1030900) were obtained from PlasmoDB. The wildtype Pfs28 sequence was used, but the Pfs25 construct used for biophysical studies contained three N-linked glycosylation sites at residues 91, 144, and 166 that were mutated from asparagine to glutamine. Single chain variable fragments (scFvs) of mAbs were designed by fusing the V_H region of each mAb to its paired V_L region by a (GGGGS)₄ linker.
All constructs were cloned into the pHLsec plasmid with a C-terminus hexa-histidine tag and transiently expressed in Expi293 cells following manufacturer protocol (Thermo Fisher Scientific, Waltham, MA) as secreted protein then harvested four to five days after transfection. After centrifugation, the supernatant was loaded on Ni Sepharose Excel resin (Cytiva, Marlborough, MA) and washed with 10 column volumes of wash buffer (25 mM Tris pH 7.4, 300 mM NaCl, 30 mM imidazole). Recombinant protein was eluted with five column volumes of elution buffer (25 mM Tris pH 7.4, 300 mM NaCl, 150 mM imidazole) and concentrated using an Amicon Ultra Centrifugal filter with 10 kDa molecular weight cutoff (Millipore Sigma, Burlington, MA). Concentrated eluate was purified by size exclusion chromatography using a Superdex 75 Increase 10/300 GL column (Cytiva, Marlborough, MA) equilibrated in 20 mM Tris pH 8.0, 100 mM NaCl.