Immunoblotting Protocol for Prion Protein Analysis

SDS-PAGE was performed with 10-well precast 1 mm 12% BisTris NuPAGE gels (NuPAGE gel electrophoresis system with MOPS buffer; Invitrogen, Breda, The Netherlands). Molecular weight markers used were MagicMark and SeeBlue Mark12 (Invitrogen). Sample volumes applied varied between 10 to 20 μl per lane, or 0.5 – 10 mg tissue equivalents (TE)/lane. Electrotransfer onto polyvinylidene difluoride membranes (PVDF, Immobilon-P; Millipore, Bedford, Mass.) and immunostaining were performed according to established procedures [67 (link), 68 (link)]. After electrotransfer, blots were blocked for 30 min with 5%skim milk protein in antibody incubation solution (25 mM Tris-HCl, 0.15 M NaCl, 2.7 mM KCl, 0.05% Tween20 at pH7.4). Primary antibodies were used at concentrations between 0.2–2 μg IgG/ml in antibody incubation solution. Secondary antibody used was rabbit anti-mouse immunoglobulinG conjugated to alkaline phosphatase (Dako, Glostrup, Denmark). Signal was developed with CDPStar by following the supplier's instructions (Tropix, Bedford, Mass.) and were recorded on photographic film, usually with exposure times between 1–45 min (Hyperfilm ECL; Amersham, Buckinghamshire, United Kingdom). Molecular weights were determined according to a method described [69 (link)]. To estimate glycoprofiles of PrP^res i.e. the relative proportions of di-, mono-, and aglycosyl fraction, films were recorded with an Agfa Duoscan T200XL scanner and further processed with GelPro software (MediaCybernetics, Silver Spring, MD) from which calculation of mutual densities of the three protein bands was possible. In experiments to compare the relative affinity for ovine PrP^res, antibodies were applied in concentration series on PVDFstrips from blots transferred from single well gels run with ovine scrapie infected brain stem homogenates varying between 1.25–20 mg tissue equivalents (TE).