Superose 6 increase 10 300 gl column

The Qβ-based VLPs (Qβ, QβApoB, QβCETP, and QβPCSK9) were characterized as previously described^{[29 (link)]} using fast protein liquid chromatography (FPLC), transmission electron microscopy (TEM), dynamic light scattering (DLS), sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE), and agarose gel electrophoresis. The Qβ VLPs concentration was determined by measuring the total protein using a Pierce BCA assay kit (Thermo Fisher Scientific). FPLC was performed using an AKTA-FPLC 900 system fitted with Superose 6 Increase 10/300 GL columns (GE Healthcare) using PBS as the mobile phase at flow rate 0.5 mL/min. TEM images were acquired on a FEI Tecnai Spirit G2 Bio TWIN transmission electron microscope. Samples were mounted on 400-mesh hexagonal copper grids and stained with 2% (v/v) uranyl acetate. DLS was carried out on a Malvern Instruments Zetasizer Nano at 25 °C in plastic disposal cuvettes. SDS-PAGE was performed under reducing conditions on NuPAGE 12% Bis-Tris protein gels [Thermo Fisher Scientific] at 120 mV for 35 min. and stained with Coomassie Brilliant Blue. Agarose gels (0.8% (w/v) in TAE buffer) were pre-stained with Gelred™ Nucleic Acid Gel Stain [GoldBio] and samples ran under non-reducing conditions at 100 mV for 30min. The gel images were acquired using the ProteinSimple FluorChem R imaging system.

The VLPs were characterized as previously described^{27 (link),28 (link),29 (link)}. The VLP concentration was determined using a Pierce BCA assay kit (Thermo Fisher Scientific). To confirm hybrid QβS100A9 assembly and peptide display, 10 μg of QβS100A9 particles was analyzed by SDS-PAGE under reducing conditions on NuPAGE 12% Bis-Tris protein gels (Thermo Fisher Scientific) stained with GelCode Blue Safe protein stain (Thermo Fisher Scientific). The gel images were acquired using the ProteinSimple FluorChem R imaging system, and densitometry was used to determine the number of peptides displayed per hybrid QβS100A9 VLP. The integrity of VLPs was confirmed by TEM using a FEI Tecnai Spirit G2 BioTWIN instrument to examine samples stained with 2% uranyl acetate. FPLC was carried out using an AKTA-FPLC 900 system fitted with Superose 6 Increase 10/300 GL columns (GE Healthcare) using PBS as the mobile phase. Particle size was confirmed by DLS using a Malvern Instruments Zetasizer Nano at 25 °C and plastic disposable cuvettes.

SEC-MALS analyses were used to determine the absolute molar masses of the protein complexes. The TSKgel G4000SW_XL (Tosoh Biosciences) and the Superose 6 Increase 10/300 GL columns (GE Healthcare) were calibrated with at least 2 column volume of the gel filtration buffer (25mM PIPES, pH 7.0, 200 mM NaCl, 1mM MgCl₂, 1mM EGTA, 1mM DTT). The columns were inline with a variable UV-absorbance detector (Agilent 1260 Infinity series) and a DAWN8+ MALS detector (Wyatt Technology). Molar masses were calculated with ASTRA 6 software (Wyatt Technology) with the dn/dc value set to 0.185 ml/g. Bovine serum albumin (BSA) was used as a calibration standard.

The concentration of VLPs was measured using a Pierce BCA assay kit (Thermo Fisher Scientific) as per the manufacturer’s protocols. VLPs were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), fast protein liquid chromatography (FPLC), dynamic light scattering (DLS), and transmission electron microscopy (TEM). SDS-PAGE was carried out using NuPAGE 12% Bis–Tris protein gels (Thermo Fisher Scientific) for 37 min at 200 V. Images were acquired using the FluorChem R imaging system (ProteinSimple) after staining the gel with Coomassie Brilliant Blue (Fisher Scientific). FPLC was performed using 300 μL of ~1 mg/mL VLP solution by an AKTA-FPLC 900 system fitted with the Superose 6 Increase 10/300 GL columns (GE Healthcare); PBS (pH 7.4) served as the mobile phase at a flow rate of 0.4 mL/min. DLS measurements were done with 100 μL of 0.1 mg/mL VLP solution using a Malvern Instruments Zetasizer Nano at 25 °C in plastic disposal cuvettes. The size was calculated from the weighted average of the number distribution. TEM was performed on an FEI Tecnai Spirit G2 Bio TWIN transmission electron microscope. The samples were prepared by mounting 0.2 mg/mL VLPs on the 400-mesh hexagonal copper grids and stained with 2% (w/v) uranyl acetate.