Ecl blotting detection reagent

After amplification, protease-resistant prion protein was detected by western blot as described previously^{61 (link)}. After proteinase K digestion (200 µg/mL) for 60 min at 45 °C and denaturation at 100 °C in SDS–PAGE denaturing buffer, samples were run on 12% polyacrylamide gel electrophoresis, before being electro-transferred onto PVDF membrane. Western blot (using the SNAP ID system, Millipore) was performed using 3F4 (mAb 3F4, epitope 109–112 of human PrP—Ozyme, France), 12B2 (mAb 12B2, epitope amino acid residues 89–93 of human PrP—Wageningen Bioveterinary, Netherlands), 9A2 (mAb 9A2, epitope amino acid residues 99–101—Wageningen Bioveterinary, Netherlands) or 6D11 (mAb 6D11, epitope 93–109 of human PrP sequence—Ozyme, France), and anti-mouse IgG peroxidase-linked secondary antibody (GE Healthcare, UK) linked to a chemiluminescent reaction (ECL blotting detection reagent, GE Healthcare, France), and imaged using films except for Fig. 3c using the imaging system Fusion FX7 (Vilber, France). The detection limit was determined visually after a maximum time exposure of 30 min and as result the dilution scored positive when the three characteristic PrP^res bands were observed.

A volume of 20 μl of each sample, prediluted or not, was incubated at 45°C with proteinase K (200 μg/ml) for 1 h before denaturation at 100°C in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer. Samples were run on 12% NUPAGE gels and electrotransferred onto polyvinylidene difluoride (PVDF) membranes. Western blots (using the SNAP ID system [Millipore]) were performed using 9A2 anti-PrP monoclonal antibodies (Wageningen, Netherlands), and an anti-mouse IgG peroxidase-linked secondary antibody (GE Healthcare, UK) linked to a chemiluminescent reaction (ECL blotting detection reagent, GE Healthcare Life Sciences, Amersham, France). For the confirmation of TSE diagnosis in mice, PrP^TSE was detected following the protocol described previously using Sha31 antibody.

Methods were performed as described previously [26 (link)]. Briefly, 20 μl of each amplified product was incubated at 45°C with PK (200 μg/ml) for 1 hr before denaturation at 100°C in SDS-PAGE sample buffer. Samples were run on 12% NUPAGE gels and electrotransferred onto PVDF membrane. Western blot (using the SNAP ID system, Millipore) was performed using 3F4 or 6D11 anti-PrP monoclonal antibodies for human and sheep prion detection, respectively (Eurogentec, 49001 Angers, France), and anti-mouse IgG peroxidase-linked secondary antibody (GE Healthcare, UK) linked to a chemiluminescent reaction (ECL blotting detection reagent, GE Healthcare life sciences, Amersham, France).