Western Blot Analysis of Prion Protein

Samples were run on 12% or 14% Tris-glycine gels using a Bio-Rad system and transferred to polyvinyl difluoride (PVDF; Millipore) membranes. Blots were immediately incubated with PrP antibody Sha31 (Spi-Bio Inc; 1/30,000 in 0.5% TBST) or were blocked with 5% skim milk in 0.1% TBST for 1 h at room temperature and incubated with primary antibody at 4°C overnight (PrP antibodies: 9A2 1/4,000, 12B2 1/8,500, SAF83 (Cayman), VRQ61 1/5,000, and 2D6 1/10,000, 1A6 1/5,000). 9A2 and 12B2 were gifts from Dr. Jan Langeveld (Langeveld et al, 2006 (link)), VRQ61 and 2D6 from Dr. Human Rezaei (Moudjou et al, 2004 (link)), and 1A6 was produced in-house. Membranes were subsequently incubated with secondary antibody at 1/10,000 (Bio-Rad) for 2 h at room temperature and visualized using ECL (Pierce). After stripping, membranes were incubated in anti-actin primary antibody (Sigma; 1/5,000).