Superdex 200 hr beads

Gel filtration, also called size exclusion chromatography, was performed as previously described [17 (link),18 ]. In brief, Superdex 200 HR beads (GE, Healthcare Biosciences, Pittsburgh, PA, USA) in a 1 × 30 cm column were used to determine the oligomeric state of PrP^Sc. Chromatography was performed in an FPLC (Fast protein liquid chromatography) system (GE, Healthcare Biosciences, Pittsburgh, PA, USA) at a flow rate of 300 μl/min, Fractions of 300 μl were collected. The molecular weight (MW) of the PrP complexes recovered in different FPLC fractions was determined using a calibration curve generated with the gel filtration of molecular mass markers (Sigma, St. Louis, MI) including Dextran blue (2,000 kDa), thyroglobulin (669 kDa), apoferritin (443 kDa), β-amylase (200 kDa), alcohol dehydrogenase (150 kDa), albumin (66 kDa), and carbonic anhydrase (29 kDa). These standards were loaded independently at the concentrations recommended by Sigma in 200 μl sample volumes. To determine the distribution of PrP in FPLC fractions, 150 μl from each fraction was concentrated by incubating with 4-fold pre-chilled methanol at −20°C for 2 h followed by centrifugation at 16,000 × g for 30 min at 4°C. After discarding the supernatant, the pellet from each fraction was re-suspended in 20 μl of sample buffer and was analyzed by Western blotting.