Size fractionation chromatography of M1000 brain homogenates prepared from different time points of the disease progression to the terminal stage.

(A, C, E, G) Representative anti-PrP immunoblots of fractions collected after size exclusion chromatography and solubilization of 10% (w/v) crude M1000 brain homogenates from 30% (A), 50% (C), 70% (E), and 100% of the terminal stage of disease (G). The fractions were immunoblotted before (top panel) and after (bottom panel) mild proteinase K treatment (PK; 5μg/mL incubated at 37°C for an hour). Since all 40 fractions could not be loaded in one 26-well gel, two 26-well gels were used for the immunoblotting of fractions from one size exclusion experiment wherein the last fraction on the first gel was also loaded as the first lane on the second gel, thereby allowing the levels of PrP on the second blot to be normalized to those on the first blot. The PrP species were probed by 03R19 antibody. Molecular weight markers are provided at left. (B, D, F) Densitometric analysis of total PrP in fractions from 30% (B; n = 3), 50% (D; n = 3), 70% (F; n = 3) of the terminal stage of disease. (H) Densitometric analysis of total PrP (PK-) and PrPSc (PK+) in fractions from the terminal stage of the disease (100%; n = 3). (B) Densitometric analysis of the immunoblots of fractions from NBH (data were extracted from our previous publication Fig 3A [31]) is superimposed over the results of M1000 fractions from 30% of the TSD to enhance comparison of PrP species in sham-inoculated mice to those at different stages of prion disease. All data are displayed as mean ± SEM.