Superdex 200 5 150 column

DNA encoding for residues 24–167 of the extracellular portion of human PD-1 (UniProt Q15116) with a C-terminus histidine tag or a corresponding PD-1 N58Q mutant was cloned into pcDNA3.4 by GenScript. Expi293 cells were transiently transfected with plasmid DNA mixed with PEI (Polysciences) and incubated for 5 d. For the expression of non-fucosylated PD-1, Expi293 cells were grown in the presence of 0.6 mM 2FPF. PD-1 proteins were purified by affinity chromatography using Ni Sepharose Excel resin (Cytiva). PD-10 columns (Cytiva) were used to exchange the buffer to PBS (Corning). Protein quality and purity were assessed by SDS–PAGE (Bio-Rad) and size-exclusion chromatography using a Superdex 200 5/150 column (Cytiva) with ACQUITY UPLC (Waters). The protein concentration was determined using a NanoDrop spectrophotometer.

50 μl samples at 10 μM concentration were loaded onto a Superose 6 5/150 (for the full length protein) analytical size exclusion column (Cytiva) equilibrated in SEC-MALS buffer (30 mM HEPES pH 6.8, 300 mM NaCl, 1 mM MgCl2, 5 μM ZnCl₂, 1 mM TCEP) attached to a 1260 Infinity II LC System (Agilent). MALS was carried out using a Wyatt DAWN detector attached in line with the size exclusion column. Mer3 fragments were analysed on Superdex 200 5/150 column (Cytiva) equilibrated in SEC-MALS2 buffer (50 mM HEPES pH 7.5, 300 mM NaCl, 1 mM TCEP).

100 μL Rpd3S sample (40 μM) was loaded onto a Superdex 200 5/150 column (Cytiva) with a buffer containing 20 mM Tris-HCl pH 7.5, 150 mM NaCl. A static light-scattering detector and a differential refractive index detector (Wyatt) were connected to the analytical gel filtration chromatography system. Data were analyzed with ASTRA7 provided by Wyatt.