Infectivity assays of fractions from the other 1.5-h gradients showed that the vast majority of infectious particles again migrated to the bottom of the tube, whereas the majority of PrP remained in the top, as shown here for pooled fractions of a gradient loaded with unsonicated supernatant. The left panel shows the %PrP in each fraction, and the right panel shows aliquots of the same samples with equivalent cell equivalents applied to GT1 indicator cells for the infectivity assay; the average of four assay points (TC-ID) are shown beneath each GT1 lane. This blot is a representative sample from GT1 cells assayed at passage 3. The bottom fractions (>30% sucrose) contain virtually all the applied infectivity, whereas the top 8% sucrose fraction with high PrP contained <3% of the TC-ID; 108% of the loaded infectivity was recovered in these gradient fractions.

Streamlined and simplified step gradient for high recovery of infectivity with very low protein background. Supernatant lysates were sonicated in 1% sarkosyl, clarified at 10,000 g × 20 min to generate p10 and su10, and the su10 was loaded on a three-step gradient (9.3 mL of su10 in 5% sucrose with 10⁸ cell equivalents was layered on 2 mL of a 10%, 0.5 mL of a 15%, and 0.1 mL of a 60% sucrose step), and spun for 1.25 h as above. Panel A shows the PrP blot, panel B shows the gold-stained blot, and panel C graphs the quantitative recovery of PrP (dark grey bars), PrP-res (light grey bars), and infectivity (black bars) in each fraction. The low-volume 60% sucrose step concentrates 75% of the infectivity and is highly purified with respect to starting protein. A few proteins, as at arrow, are also selectively enriched with little background.

The graphs shows the quantitative recovery of PrP, PrP-res, protein, and infectivity in two representative gradients centrifuged for 2.5 h. Gradient A is from Fig. 1, and gradient B is a repeat preparation in which 1% sarkosyl was added in the supernatant, and was 0.1% in the sucrose steps. PrP in each fraction is shown by dark grey bars, and the percentage PrP-res of that fraction by light gray bars. The PrP and PrP-res profiles are the same in both gradients, with quantitative recovery of PrP in each gradient (denoted by arrow). Standard protein solution assays of both gradients in panel C, in accord with gold blot analyses, show that the vast majority of protein stays in the top fractions. Panel D shows the infectious titers (TC-ID) in the supernatant, and in each gradient fraction of A and B (without and with sarkosyl, respectively). Virtually all of the infectivity migrates to the bottom 60% sucrose fraction in both gradients, and no pellets were visible. The percentage of the loaded infectivity recovered from the gradient fractions is indicated by the arrows. Panel E shows one of the four GT1 assay cultures (passage 5 for gradient A) analyzed for infectivity; titers were calculated from the %PrP-res produced in infected GT1 indicator cells (8). The highest infectivity corresponds to the highest percentage of PrP-res that was evoked in GT1 indicator cells. Dilutions of sample fractions containing equal cell equivalents were applied to GT1 cells for comparison of fraction infectivity, and brain is a parallel 22L control homogenate with a known LD₅₀ in mice assayed in parallel. Application of uninfected N2a cells at high cell equivalents gave no PrP-res signal, and microcon concentrated fractions were comparable to untreated fractions (data not shown).

Sonicated supernatant samples loaded on more concentrated sucrose steps were centrifuged for a reduced time of 1.25 h (259,000 g-h). The top gradient (A) was loaded with 5 × 10⁷ cell equivalents, and the PrP Western blot with corresponding gold stain are shown. Unlike the 2.5-h gradients in Fig. 2, substantially more PrP (69%) remains at the top of the gradient (8% sucrose layer); 97% of the loaded PrP was recovered from this gradient. Even without proteinase K digestion, smaller PrP-res bands (as at arrow) are more prominent in the lower two gradient fractions that together contain 20% of the loaded PrP, as well as reduced protein by gold staining. Gradients B and C show PrP and PrP-res in a repeat gradient loaded with 10⁸ cell equivalents in which the relative %PrP-res was rigorously quantified. Panel B shows the undigested PrP samples, and panel C shows aliquots of the same samples digested with proteinase K. Again, the majority of total PrP remains at the top of the gradient, with less than 10% in the bottom two fractions. Lane loads and the percentage of PrP and PrP-res in each fraction are indicated. In each of the gradients ~7 mL of sonicated supernatant in 8% sucrose was loaded on top of a step gradient made with (from top to bottom) 1 mL, 2 mL, 1 mL, 1 mL, and 0.3 mL, respectively, of the sucrose concentrations indicated. As in previous gradients, the entire supernatant was collected as a single fraction (up to the interface), and ~0.5-mL fractions collected from the 20% sucrose step downwards.

Western blot of a N2a-22L cell lysate (LY), nuclear pellet (p), and cytoplasmic supernatant (su) fraction centrifuged on a sucrose step gradient for 2.5 h (453,000 g-h). The top panel shows PrP detected with antibody M20 before and after proteinase K digestion (PK − and +, respectively). The bottom panel shows gold-stained total proteins in the PK− lanes (stained after PrP detection). Loads on each lane were proportional and standardized for comparable cell equivalents (e.g., 10 μL of a 5-mL 5% sucrose layer is equivalent to 1 μL of a 0.5-mL 15% sucrose step layer). Negligible amounts of PrP or PrP-res are lost in the nuclear pellet (loaded with 20× and 30× of the starting lyate), whereas essentially all PrP and PrP-res are recovered in the supernatant. Total gold stained protein shows almost all the loaded lysate protein is recovered in the supernatant, with little protein in the nuclear pellet (p); this pellet showed high DAPI fluorescence, and also revealed a highly selective concentration of only a few protein bands (e.g., at arrow), while the supernatant contained little nuclear DNA (nucleic acid sequences in infectious fractions will be reported separately). To test if microcon concentration with buffer washing could be used to reduce detergent and sucrose concentrations without losses, aliquots of the microcon-filtered supernatant and several sucrose steps were compared (* samples); the PrP, PrP-res, and protein in both were comparable. Note that the bottom-most 60% sucrose fraction (loaded at 8× the supernatant) has very little protein, but a relatively high concentration of PrP and PrP-res. This gradient was loaded with 1.2 × 10⁸ cell equivalents (CE) of supernatant. The cytoplasmic supernatant was brought to ~8.8 mL in 5% sucrose, and loaded onto a step gradient with 1 mL of 10%, 1 mL of 15%, 0.5 mL of 30%, and 0.1 mL of 60% sucrose in TE buffer.