Criterion xt bis tris precast gel

The eluted proteins were precipitated with trichloroacetic acid (TCA) to enrich them for subsequent analyses. Sodium deoxycholate was added to a final concentration of 0.02%. Samples were mixed, placed on ice and supplemented with TCA to a final concentration of 10%, and then incubated on ice for 1 h. Precipitated proteins were pelleted by centrifugation (10 min, 16000 × g, 4°C), washed with 100% ice-cold acetone by incubating them on ice for 15 min and subsequent centrifugation (10 min, 16000 × g, 4°C). Washing was repeated once; the pellet was air-dried and boiled in SDS-PAGE buffer (7.5 μl 4x XT Sample Buffer, 1.5 μl 20x XT Reducing Agent, 1% SDS, ad 30 μl H2O) for 5 min at 95°C. The samples were separated on Criterion XT Bis-Tris Precast Gels (125 V for 1.5 h in XT MES Running Buffer) (all from Bio-Rad Laboratories GmbH). PageRuler Plus Prestained Protein Ladder (Thermo Fisher Scientific) was used as a size standard. For a subsequent Coomassie staining, the gel was equilibrated in H₂O for 5 min, incubated in 100 ml acetic acid/ethanol/H₂O (10:40:50) for 1 h and subsequently rehydrated with H₂O (three times, 10 min). The gel was stained with PageBlue^TM Protein Staining Solution (Thermo Fisher Scientific) overnight and repeatedly washed with H₂O for destaining.

Before the centrifugation step of the homogenization procedure, PrP^Sc was extracted with the BioRad TsSeE purification and detection kit (Marnes-la-Coquette, France) and digested with 200 μg/ml proteinase K (Euromedex, Mundolsheim, France) at 37°C for 10 min. After denaturation in Laemmli buffer at 100°C for 5 min, samples were run on 12% Criterion™ XT Bis-Tris Precast Gels (Bio-Rad), electrotransferred to nitrocellulose membranes, and probed with the biotinylated anti-PrP monoclonal antibody Sha31, as previously described [46 (link)]. Immunoreactivity was visualized by chemiluminescence (GE Healthcare, Orsay, France).

The kidney samples were lysed in RIPA Buffer (Thermo Scientific, Rockford, IL). Protein concentration was determined by the bicinchoninic acid (BCA) protein assay (Thermo Scientific, Rockford, IL). Twenty μg protein was resolved on 4–12% Criterion XT Bis-Tris Precast gels (BioRad, Hercules, CA) and transferred to nitrocellulose membrane to detect HNE or to Polyvinylidene Difluoride (PVDF) membrane to detect NT. The primary NT antibody was applied at 1.3 μg/mL and the primary HNE antibody at 0.3 μg/mL. The secondary antibody (peroxidase conjugated goat anti-mouse, PerkinElmer, Santa Clara, CA) was applied at 0.25 μg/mL. Blots were incubated in enhanced chemiluminescence substrate, Supersignal West Pico Chemiluminescent Substrate (Thermo Scientific, Rockford, IL), and were exposed to photographic film. After stripping membrane with Restore Western Blot Stripping Buffer (Thermo Scientific, Rockford, IL), as a loading control, peroxidase conjugated anti-actin (AC-15 Abcam, Cambridge, MA) was applied at 70 ng/mL concentration in blocking buffer for 1 h at room temperature.

For SDS-PAGE analysis, the pellets containing the cytoplasmic contents were treated with CelLytic™ B Cell Lysis Reagent (Sigma-Aldrich). An additional 50 units/mL benzonase nuclease (Sigma-Aldrich) was used for all samples. The samples were incubated for 30 min at room temperature with shaking (100 rpm). Dilutions (5x) of the samples were done with XT MES Running Buffer (Bio-Rad). Each of the samples was combined with XT Sample Buffer 2X (Bio-Rad) and with XT Reducing Agent (Bio-Rad) and incubated at 95 °C for 5 min. Samples (10 μL, 10x diluted) and a ladder (Precision Plus Protein™ Dual Color Standards, Bio-Rad, 5 μL) were loaded on SDS-gel (Criterion™ XT Bis-Tris Precast Gels, 12%, Bio-Rad) for gel electrophoresis (200 V, 45 min), which after end of run was stained with InstantBlue™ Coomassie Protein Stain (Expedeon).

Protein content of samples for SDS-PAGE was analyzed using a Pierce 660 nm protein assay reagent (Thermo Scientific) following the manufacturer’s recommendations with a standard curve from 0 to 2000 µg mL⁻¹ bovine serum albumin. Samples were incubated for 5 min at 95 °C with sample loading buffer [containing 2% SDS and 0.1 M dithiothreitol (DTT)] prior to separation by SDS-PAGE. Proteins were separated on 12% Criterion XT bis–tris pre-cast gels (Bio-Rad Laboratories) in 2-morpholinoethanesulphonic acid (MES) running buffer at 180 V. Afterwards, the gels were either stained with Coomassie Blue, Oriole Fluorescent Gel Stain (Bio-Rad Laboratories) (protocol according to manufacturer’s instructions) or immunoblotted (see “Immunoblot analysis”).

Fibrils were treated with proteinase K (proteinase K to PrP ratio of 1:100) at 37 °C for 1 h. 5 μg of PrP fibrils were thus digested using 0.05 μg of PK, and the digestion reaction was stopped with 2 mM phenylmethylsulfonyl fluoride. Samples were heated in denaturing sample buffer (60 mM Tris– HCl, 2% SDS, 5% β -mercaptoethanol, 2.25 M urea) at 95 °C for 10 min and separated on 12% Criterion™ XT Bis-Tris Precast Gels (Bio-Rad) followed by silver staining. Quantification of protein-band intensities was performed by densitometric analysis using Image Lab software (Biorad).