Bm chemiluminescent substrate kit

Brain homogenates (10% weight/volume) were prepared in NP40 lysis buffer (1% NP40, 0.5% sodium deoxycholate, 150 mM NaCl, 50 mM TrisHCL [pH 7.5]) and incubated at 37 °C for 1 h with 20 µg/ml PK. Digestions were halted by addition of 1 mM phenylmethylsulfonyl fluoride. Samples were then subjected to electrophoresis through 12% Tris-glycine polyacrylamide gels (Nupage, Life Technologies) and transferred to PVDF membranes by semi-dry blotting. PrP was detected using anti-mouse PrP-specific mAb 7A12 (Yin et al., 2007 (link)) followed by horseradish peroxidase-conjugated goat anti-mouse antibody (Jackson Immunoresearch) and visualised chemiluminescence (BM Chemiluminescent substrate kit, Roche, Burgess Hill, UK).

Brains samples were homogenised in NP-40 lysis buffer (1% NP-40, 0.5% sodium deoxycholate, 150 mM NaCl, 50 mM Tris-HCL [pH 7.5]) at 10% weight/volume, and subsequently treated at 37 °C for 1 h with proteinase K (20 µg/ml). Samples were then separated by electrophoresis and electroblotted onto polyvinylidene difluoride membranes as described previously^{42 (link)}. PrP was detected using anti-mouse PrP mAb (clone 7A12; Yin et al., 2007), followed by HRP-conjugated anti-mouse antibody (Jackson Immunoresearch, Ely, UK) and visualised with BM Chemiluminescent substrate kit (Roche, Burgess Hill, UK). An uncropped image of the immunoblot is provided in Supplementary Fig. S1.

Brain homogenates (10% wt/vol) were prepared in NP40 lysis buffer (1% NP40, 0.5% sodium deoxycholate, 150 mM NaCl, 50 mM TrisHCl [pH 7.5]). For the detection of PrP^Sc a sample of homogenate was incubated at 37°C for 1 h with 20 μg/ml proteinase K (PK) and digestion halted by addition of 1 mM phenylmethylsulfonyl fluoride. Samples were denatured at 98°C for 15 min in 1× SDS sample buffer (Life Technologies) and separated via electrophoresis through 12% Tris‐glycine polyacrylamide gels (Nupage, Life Technologies) and transferred to polyvinylidene difluoride PVDF membranes by semi‐dry electroblotting. Primary antibodies (Table 1) were detected by horseradish peroxidase‐conjugated goat anti‐species specific antibody (Jackson Immunoresearch) and visualized via chemiluminescence (BM Chemiluminescent substrate kit, Roche, Burgess Hill, UK) as described previously (Bradford et al., 2017 (link)).