Nupage 12 bis tris protein gel

E. coli strains containing the pET3a-based plasmids were incubated and induced as for the in vivo cyclase assay. After 5 h induction cells were harvested and resuspended in 50 mM Tris–HCl, pH 8.0, 5 mM EDTA, supplemented with Proteinase Inhibitor Cocktail (Sigma–Aldrich), and lysed by sonication on ice (3 ×  20 s with 20 s resting between cycles). The cell lysate was clarified by centrifugation at 3000×g at 4°C for 5 min to remove unbroken cells and cell debris, followed by SDS–PAGE analysis using NuPAGE 12% Bis-Tris Protein Gels (Thermo Fisher Scientific). Protein bands were transferred to a polyvinylidene difluoride (PVDF) membrane, which was probed with specific primary antibodies raised against Arabidopsis CHL27 (Agrisera), YCF54 (Agrisera) and Synechocystis Ycf54 [5 (link)], and then with a secondary anti-rabbit antibody conjugated with horseradish peroxidase (Sigma–Aldrich). Chemiluminescent signal was developed using the WESTAR SUN enhanced chemiluminescence substrate (Cyanagen) and detected by an Amersham Imager 600 (GE Healthcare).

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 VLPs were characterized by fast protein liquid chromatography (FPLC), transmission electron microscopy (TEM), dynamic light scattering (DLS), and sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). FPLC was performed using an AKTA-FPLC 900 system fitted with Superose 6 Increase 10/300 GL columns (GE Healthcare), using PBS (pH 7.4) as the mobile phase at a flow rate of 0.5 mL/min. TEM images were acquired on an FEI Tecnai Spirit G2 Bio TWIN transmission electron microscope. Samples were mounted on 400-mesh hexagonal copper grids and stained with 2% uranyl acetate. DLS was carried out on a Malvern Instruments Zetasizer Nano at 25 °C, in plastic disposal cuvettes. SDS-PAGE was performed on NuPAGE 12% Bis-Tris protein gels (Thermo Fisher Scientific) at 150 mV for 75 min. The gels were stained with Coomassie Brilliant Blue and images were acquired using the ProteinSimple FluorChem R imaging system.

Tg2576 mouse brains were lysed in RIPA buffer (Sigma-Aldrich) with protease and phosphatase inhibitors cocktail (Thermo Fisher Scientific). The lysate was incubated with the LAMP1 cytoplasmic or normal rabbit IgG antibodies, and then precipitated using Protein G Dynabeads (Thermo Fisher Scientific). Immunoprecipitated proteins were eluted in the elution buffer (50 mM Glycine pH2.8, LDS sample buffer, Reducing Agent; Thermo Fisher Scientific) by boiling for 3 min at 95 °C. Then, the eluted samples were subjected to SDS–PAGE on NuPAGE 12% Bis-Tris Protein gels (Thermo Fisher Scientific). The Bio-Rad Wet electroblotting system (Bio-Rad) was used to transfer proteins from the gels to nitrocellulose membranes (Thermo Fisher Scientific). The detection was performed by immunoblotting with specific primary and corresponding fluorophore conjugated secondary antibodies and their fluorescent signals were detected using the LI-COR Odyssey CLx scanner (LI-COR Biosciences (Lincoln, NE, USA)).