Bolt bis tris plus

Fab size and homogeneity were assessed by Sodium Dodecyl Sulfate - PolyAcrylamide Gel Electrophoresis (SDS-PAGE) and SEC. For SDS-PAGE, 5 μg protein/lane was loaded on a 4%–12% Bolt Bis-Tris Plus gel (Thermo Fisher Scientific) in reducing VS non-reducing conditions, and run at 200 V in 3-Morpholinopropane-1-sulfonic acid (MOPS) buffer. Bands were visualized with Coomassie staining (Expedeon), and the size of the fragments evaluated by running a protein standard ladder (Thermo Fisher Scientific). For SEC, 50 μg protein was loaded on a Superdex 200 increase 10/300 column using a 100 μL loop, and run at 0.5 mL/min using an Äkta Pure system (GE Healthcare). Fab peaks were analyzed with the provided Unicorn 7.0.2 software. The size of the fragments was estimated with the help of a linear regression calculated by running a mix of proteins with known molecular weight (BioRad) on the same column.

Ninety percent of the neat elution from each GFP pull-down was resolved by SDS–PAGE (4–12% Bolt Bis–Tris Plus precast gels, Invitrogen) and visualized with Colloidal Blue (Invitrogen) following manufacturer’s instructions. Aiming to isolate mono- and poly-ubiquitinated DDI1, each gel lane was excised into two slices above the clear band of approximately 75 KDa that corresponds to non-modified DDI1 (Figure 1). Subsequently, each gel lane was chopped, and proteins were subjected to in-gel digestion protocol using trypsin as described before (Osinalde et al., 2015 (link)). Briefly, gel pieces, previously dehydrated with acetonitrile (ACN), were reduced with 10 mM dithiothreitol (DTT), alkylated with 55 mM chloroacetamide, and finally rehydratated in 12.5 ng/ml trypsin and incubated overnight at 37°C. The following day, resulting tryptic peptides were extracted from the gel by serial incubation with 100% ACN and 1% trifluoroacetic acid in 30% ACN. Finally, prior to MS analysis, peptide solutions were dried down in a SpeedVac centrifuge (Thermo Scientific).

LPS was isolated and analyzed as previously described (70 (link)). Briefly, bacteria were grown at 37°C to an OD₆₅₀ of ~0.5, and the equivalent of OD₆₅₀ of 1 was pelleted and resuspended in Laemmli SDS-PAGE sample buffer (69 (link)). Samples were then boiled for 10 min, cooled to room temperature, and treated with 25 μg proteinase K for 1 h at 55°C. LPS was visualized by (4 to 12%) SDS-PAGE (Bolt Bis-Tris Plus; Invitrogen) and subsequent staining of the LPS with silver stain as follows. The gels were fixed in 40% isopropanol and 5% acetic acid, oxidized with 0.7% periodic acid, stained with 20% silver nitrate, and developed using 50 μg/ml citric acid.

Brain extracts containing either soluble or insoluble α-synuclein were supplemented with LDS loading buffer, heated at 100 °C for 10 min and loaded into 12% Bolt Bis-Tris Plus gels (Invitrogen). Proteins were separated by means of SDS-PAGE and then transferred onto Polyvinylidene difluoride (PVDF) membranes (Immobilon-P, Millipore) and incubated with 5% (weight/volume) non-fat dry milk (prepared in Tris-HCl with 0.05% Tween-20) for 1 h at room temperature under shaking. PVDF membranes were incubated with primary antibody to α-synuclein (polyclonal AS08 358 Agrisera: epitopes 1–15) overnight at 4 °C under shaking.
RT-QuIC products were digested with proteinase K (see section PK digestions) and analyzed by Western blot following the same procedures described above. In this case, PVDF membranes were incubated with primary antibodies directed against different epitopes of α-synuclein (monoclonal 4D6 Invitrogen: epitopes 124–134 [65 (link)]; polyclonal AB5038 EMD Millipore: epitopes 111–131 [66 (link)]; monoclonal 5C2 Novus Biologicals: epitopes 61–95 [67 (link)]; polyclonal AS08 358 Agrisera: epitopes 1–15 [68 (link)]) overnight at 4 °C under shaking. Membranes were incubated with appropriate secondary antibodies conjugated with horseradish peroxidase (GE) and developed with chemiluminescent system (ECL Prime). Reactions were visualized using a G:BOX Chemi Syngene system.

Thirty-forty micrograms of lysate proteins for each sample together with the molecular weight Magic Mark (Invitrogen, Carlsbad, CA, USA) were subjected to 4–12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis separation (Bis-Tris Plus BOLT, Invitrogen) and transferred to polyvinylidene fluoride membranes (PVDF, Millipore, Burlington, MA, USA). The membranes were blocked in 5% skim milk and incubated overnight with the following specific primary antibodies: rabbit anti-p16 (N-20, sc-467, Santa Cruz Biotechnology, Dallas, TX, USA), rabbit COX-2 (160126, Cayman Chemical, Ann Arbor, MI, USA), rabbit anti-NFkB (GTX102090, GeneTex, Irvine, CA, USA), mouse anti-α smooth actin (A2547, Sigma-Aldrich Chemicals, Italy), rabbit anti-Glucose 6 Phosphate Dehydrogenase (GAPDH) (14C10, Cell Signaling) followed by the suitable HRP-conjugated secondary antibodies (Sigma-Aldrich Chemicals). All resulting immunocomplexes were visualized with an enhanced chemiluminescence ECL detection system (GE Healthcare, Milano, Italy) and quantified by ImageJ software (NIH, Bethesda, MD, USA). Each density measure was normalized by using the corresponding GAPDH level as an internal control.