Sarkosyl 20

Genomic high molecular weight DNA was isolated from 50 mg of kidney from Ie and wild type littermates. Tissue lysis was performed using 500 µL of 0.25% trypsin-EDTA (59428C, Sigma-Aldrich, MA, USA) and 5 mL of 1× red blood cell lysis buffer. Cell lysates were obtained using 3 mL lysis buffer (20 mM Tris-HCL pH7.5, 75 Mm NaCl, 50 mM EDTA pH8), 30 µL proteinase K PCR-grade (20 mg/mL) (03 115 879 001, Roche, Basel, Switzerland) and 150 µL Sarkosyl 20% (Sigma-Aldrich, MA, USA). A standard DNA isolation procedure using phenol:chloroform was performed. We used 1 µL DNAase-free RNase (Roche, Basel, Switzerland) to remove remaining RNA. DNA was purified using 1.2× Ampure XP beads. Genomic DNA was analysed for quality by electrophoresis with a 0.8% agarose gel stained with GelRed (ThermoFisher Scientific, MA, USA).

50 μL of (1) FFI-BH (10-fold concentrated by means of high-speed centrifugation), (2) FFI-BH_PMCA, and (3) brain homogenates of BvPrP-Tg407-inoculated mice were treated with 450 μL of guanidine hydrochloride (Gdn-HCl; Sigma) at different molar concentrations (1.5, 3, 4.5, and 6 M) for 2 hr at 25°C under shaking (550 rpm). Subsequently, an equal volume of sarkosyl 20% (Sigma) was added to the preparation that was incubated for 10 min with gentle shaking. Samples were centrifuged at 100,000 × g for 1 hr at 4°C. Pellets were washed with PBS and then centrifuged at high speed (100,000 × g, 30 min at 4°C). The resulting pellets were suspended in 50 μL of loading buffer (Bolt LDS Sample Buffer and DTT, ThermoScientific) and then subjected to Wb analysis as described. Membranes were immunoblotted using the 6D11 antibody. Each densitometric analysis of the resulting bands was performed (at least three times per sample) using ImageJ software.