Figure 9

RT-QuIC seeding activity of VPSPr and VV2 sCJD after sucrose density gradient separation. (a) An adjacent sample from VPSPr frontal cortex to the one analysed below [in (b)] was homogenised to 10% w/v in extraction buffer and analysed by immunoblotting using 3F4 as the primary anti-PrP monoclonal antibody either without prior PK digestion (15 μl) or following digestion with 50 μg/ml PK and centrifugal concentration (from 100 μl). The densitometric analysis of the ~19 and ~23 kDa bands versus the ~8 kDa band in this sample is superimposed on the figure. (b) Samples of SDGC fractions from VPSPr case 1 (200 μl) and sCJD VV2 (100 μl) were PK treated (50 μg/ml, 1 h) precipitated overnight with nine volumes of methanol, and analysed for PrP^res by western blotting. The positions of molecular mass markers (in kDa) are indicated on the right. The collective densitometric analysis of the ~19 and ~23 kDa bands versus the ~8 kDa bands in the SDGC-separated sample is superimposed on the figure. (c) VPSPr (case 1), sCJD VV2 and non-CJD brain homogenate were separated by sucrose density gradient centrifugation (SDGC). The gradient used was 10-60% sucrose and after centrifugation eleven fractions of increasing density were successively taken from the top of the tube (#1 = lowest density, #11 highest density). Duplicate RT-QuIC reactions were seeded with 2 μl samples of 1:10 dilutions of the fractions. The ThT relative fluorescence values plotted are the means of the duplicate reactions at 40 hours.