Protease-resistant sCJD-PrP^Sc is associated with ferritin. Clarified supernatant obtained from PK-treated CJDH (S1) was subjected to five additional rounds of ultracentrifugation, and the resulting detergent-soluble supernatant (S2-S6) and detergent-insoluble pellet (P2-P6) fractions were immunoblotted with 3F4. A, The starting material (S1) and the supernatant and pellet fractions obtained after ultracentrifugation (S2 and P2, respectively) show the N-terminally truncated PrP^Sc glycoforms migrating at 29 and 30, 22-25, and 19 kDa, respectively (lanes 1-3). After the first round of ultracentrifugation, 60% of PrP^Sc fractionates in the pellet fraction (lanes 2, 3). After subsequent rounds of ultracentrifugation, all of the PrP^Sc is detected in the pellet fractions (lanes 4-11). B, Silver staining of the P6 fraction shows PrP^Sc glycoforms of 19-30 kDa and a 20 kDa band (lane 1). After treatment with PNGase, all PrP^Sc glycoforms migrate at 19 kDa, as expected (lane 2, arrowhead), whereas the 20 kDa band remains insensitive to deglycosylation (lane 2, arrow). The slower migrating band in lane 2 (^*) is derived from the added PNGase. C, Reprobing of the membrane in A with anti-ferritin antibody shows strong immunoreaction with the 20 kDa band and minor species migrating at 25 and 30 kDa (lanes 1-11). The 20 kDa band is detected in the first three fractions (lanes 1-3) and in all subsequent pellet fractions (lanes 4-11). D, The P6 fraction from A was treated with NaCl and subjected to DEAE-cellulose chromatography. Immunoblotting of eluted proteins with 3F4 and anti-ferritin antibodies shows dissociation of ferritin from PrP^Sc at concentrations of 0.2-0.4 m NaCl (lanes 2, 3).