6H4 Methods: Western Blot
WB detection of the protease-resistant core of PrPSc
(denominated PrP 27-30)
Tissue samples were homogenized in Prionics®-Check Homogenization buffer using the PrioGENIZER homogenization system for 45 sec. at 24’000 rpm. Protease treatment was done with 2 U/ml proteinase K at 47 °C for 30 min. The protease was inactivated using Pefablock SC. For WB, all homogenates were diluted 1:1 in SDS sample buffer (Prionics), heated to 96 °C for 5 min and electrophoresed on 12% polyacrylamide gels containing SDS (using the Bio-Rad Mini-Protean System or Novex 12% NuPAGE precast gels) at 200 V for 1 h. The proteins were transferred to PVDF membranes at 150 V for 1 h at +4 °C in a transfer tank equipped with plate electrodes. At this stage, proteins bound to the PVDF membrane can be identified by staining with 0.025% (w/v) Ponceau S dissolved in 0.25% acetic acid. Nonspecific protein binding sites on the PVDF membrane were blocked by incubation in PVDF blocking buffer (Prionics) for 30–60 min at room temperature. Protease-resistant PrP was detected by incubation with monoclonal antibody (mAb) 6H4 at a dilution of 1 : 5,000 for 2 h at room temperature, or for 12-18 h at +4 °C, followed by incubation with goat anti-mouse IgG antibodies coupled to alkaline phosphatase (AP). After equilibration of the PVDF membrane in 20 mM TRIS-HCl, 1 mM MgCl2, pH 9.8 for 5 min, AP activity was visualized by application of 0.25 mM CDP-Star in 20 mM TRIS-HCl, 1 mM MgCl2, pH 9.8. The disease-specific form of PrP, PrPSc, is characterized by its partial protease resistance, i.e., the N-terminal 60- to 70-amino acids are cleaved, while the rest of the protein remains intact. This leads to a reduction in molecular mass from about 35 to 27–30 kDa.
Adapted from
Schaller O et al., (1999) Validation of a Western immunoblotting procedure for bovine PrPSc detection and its use as a rapid surveillance method for the diagnosis of bovine spongiform encephalopathy (BSE). Acta Neuropathol (1999) 98:437–443
