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 PrPres 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.