Tesee western blot kit

Western blotting was performed following a protocol based on TeSeE Western Blot kit (Bio-Rad). Briefly, medulla oblongata and mesenteric lymph node samples from sheep and spinal cord pools from diseased mice were thawed and homogenised in a detergent-containing solution. A volume of 200 µl was submitted to proteinase K digestion for 10 min, which was stopped using a β-mercaptoethanol-containing stop buffer, followed by concentration, clarification and resuspension of the remaining PrP^res in 30 µl of Laemli loading buffer. It was then subjected to SDS-PAGE electrophoresis using commercial 12% Bis-Tris gels (Bio-Rad), followed by transference to a PVDF membrane with 0.20-µm pore diameter (Bio-Rad). Immunoblot was performed by sequentially immerging the membrane in a blocking solution (0.2% BSA in PBS+Tween 0.1%), primary anti-PrP antibody Sha31 (whose epitope spans amino acids 148–155) diluted 1:8,000 in PBS+Tween 0.1%, and HRP-conjugated secondary antibody diluted 1:5,000 in PBS+Tween 0.1%. Finally, membranes were developed by incubation with a luminol-based substrate (SuperSignal West Pico Chemiluminescent Substrate).

Western blot was performed on the original isolates, some of the substrates, and the PMCA products using the TeSeE Western Blot kit (Bio-Rad) following the manufacturer’s instructions. The tested PMCA products and the isolates were subjected to a one-hour digestion with 100 µg/mL proteinase K (PK) (Sigma-Aldrich) at 37 °C. This step was omitted for the cervid substrates to detect the host PrP^C. The reaction was stopped by incubating the mixture with Laemmli sample buffer (Bio-Rad) for five minutes at 100 °C. The samples were then loaded onto Sodium Dodecyl Sulfate–Polyacrylamide gel (NuPAGE 12% Bis–Tris protein gels, Thermo-Fisher) and electrophoresed at 100 V for 55 min. The proteins were subsequently transferred to a polyvinylidene difluoride membrane using a trans-blot turbo system (Bio-Rad), and the membrane was then subjected for immunodetection. The commercial kit included the primary monoclonal antibody (mAb), Sha31, which recognizes the core epitope 148YEDRYYRE155. Additionally, the mAb P4 (Labolytic), which identifies the N-terminal epitope 93WGQGGSH99 (ovine numbering for both antibodies), was used. The results were visualized using Azure c280 (Azure Biosystems). Brains from a scrapie-positive sheep, either alone or together with a moose CWD isolate, were utilized as positive controls.

The TeSeE Western blot kit (Bio-Rad) was used following the manufacturer’s recommendations. Samples taken from the brain, sciatic nerve, brachial nerve, mesenteric lymph node, oculomotor muscle, ileum, and spleen, and WBCs obtained from Sh-BSE-challenged pigs, were analysed for the presence of PrP^res. For PMCA products, 20 μl of reaction product was mixed with 230 μl of 10% negative brain homogenate before PrP^res extraction, as previously described [26 (link)]. PrP^res detection was performed using Sha31 mAb conjugated to horseradish peroxidase (0.06 μg/ml). ECL substrate (Pierce) was used to reveal peroxidase activity.