Superose 6 pc 3.2 30 column

Freshly thawed plasma samples were diluted with phosphate-buffered saline (PBS; 1 volume plasma:5 volumes PBS). Lipoprotein particles were separated on a Shimadzu Prominence UFLC system using a Superose 6 PC 3.2/30 column (GE Healthcare Europe GmbH, Munich, Germany) with PBS as a mobile phase (at 50 μl min⁻¹). The eluent was led to a Syrris FLLEX module (Syrris, Royston, Herts, UK), which also received CHCl₃/MeOH (3:1) at 50 μl min⁻¹. Using a 250 μl loop the two phases were mixed and then separated using a Teflon membrane in the pressurized glass flow chamber of the FLLEX module. This produced both the aqueous and organic phases as separated eluents. The organic phase was mixed with 1:1 isopropanol/methanol containing 7.5 mM ammonium acetate at 150 μl min⁻¹ and led to a standard electrospray source on a LTQ Orbitrap Velos for the acquisition of mass spectra.

Analytical gel filtration chromatography was performed using the SMART system (Amersham Pharmacia, Buckinghamshire, UK). PolD (wt, ΔPIP, ΔKR, ΔPIPΔKR), PCNA, and DNA (each 5.4 μM, as a heterodimer: PolD, homotrimer: PCNA in 30 μl) were mixed and incubated for 3 min at 60 °C. The protein solutions were applied to a Superose 6 PC 3.2/30 column (GE Healthcare) and were eluted with buffer containing 50 mM Tris–HCl, pH 8.0, and 0.3 M NaCl. Aliquots (5 μl) of applied solution and aliquots (8 μl) of each fraction from the eluates were subjected to 10% SDS-PAGE, followed by Coomassie Brilliant Blue staining. The standard marker proteins, including thyroglobulin (670,000), γ-globulin (158,000), ovalbumin (44,000), and myoglobin (17,000), were also subjected to gel filtration as controls.

Fifty µl of a peak fraction of the Ni²⁺-NTA column (0.22 µm-filtrated) was applied to pre-equilibrated Superose 6 PC3.2/30 column (GE Healthcare), and run in 20 mM Tris-HCl [pH7.8], 150 mM KCl, 1 mM EDTA, 0.5 mM DTT, 10% glycerol and protease inhibitors [cOmplete^™ Protease Inhibitor Cocktail; Roche] at 40 µl/min at 4 °C. Eighty µl-fractions were collected and subjected to SDS-PAGE followed by western blotting with anti-DDDDK antibody (FLA-1, MBL Co.) to detect the C-terminal FLAG₃ tag. Molecular size standards (GE Healthcare) were chromatographed under the same condition, and retention volumes of all the standards were determined by monitoring UV absorbance at 260 nm.

For EM analysis, gB-698glyco was incubated with 1G2 Fab at a 1:1.5 molar ratio on ice for 1 h and resolved over a Superose 6 PC 3.2/30 column connected to an Akta Micro system (GE Lifesciences). For visualization, 5 μl of sample was incubated on a freshly discharged continuous carbon 400-mesh copper grid (Electron Microscopy Sciences) for 30 sec. Following incubation, the grid was moved through 5 × 75 μl droplets of a 2% (w/v) uranyl formate solution (SPI Supplies). Excess stain solution was blotted away and the grid was air-dried. EM images were collected on a Tecnai-12 Spirit (FEI) equipped with a LaB6 filament operated at 120 keV under low dose conditions using a 4 k × 4 k CCD camera (Gatan Inc.) at a nominal magnification of × 49,000 (2.15 Å per pixel). Particles were isolated from individual micrographs using the e2boxer.py procedures embedded into EMAN2 (ref. 53 (link)) and subjected to iterative two-dimensional reference-free image analysis using multivariate statistical analysis followed by multi-reference alignment in IMAGIC5 (ref. 54 (link)). To obtain a gB/1G2 three-dimensional reconstruction, ∼10,000 particles were refined against an initial model of HSV gB (PDB ID 2GUM) low pass filtered to a resolution of 60 Å using the EMAN2/Sparx software53 (link)55 (link) and fitted in Chimera56 (link).