Protein g sepharose fast flow

For the IP analysis, treated cells were collected and disrupted in IP analysis buffer (300 mM NaCl, 50 mM Tris, pH 8.0, 0.4% NP-40, 10 mM MgCl₂, 2.5 mM CaCl₂) containing protease inhibitors (Roche Diagnostics, 04693116001). Total cell extracts (1 mg per sample) were mixed with precleared protein G Sepharose^TM Fast Flow (GE Healthcare, 17-0618-01) and appropriate antibodies, followed by incubation for 4 h at 4 °C. The beads were collected by centrifugation, washed five times and resuspended in 2× SDS loading buffer and then subjected to western blotting. The protein bands were visualized using DyLight 800/DyLight 680-conjugated secondary antibodies, and the infrared fluorescence image was obtained using an Odyssey infrared imaging system (LI-CORBiosciences, Lincoln, NE, USA).

For the IP analysis, treated cells were collected and disrupted in RIPA Lysis Buffer containing protease inhibitors (Roche Diagnostics, 04693116001). The total cell extracts (1 mg per sample) were mixed with precleared protein G sepharoseTM Fast Flow (GE Healthcare, 17-0618-01) and the appropriate antibodies, followed by an incubation for 2 h at 4 °C. The beads were collected by centrifugation, washed five times, resuspended in 2 × SDS loading buffer, and analyzed by Western blotting. The protein bands were visualized using DyLight 800/DyLight 680-conjugated secondary antibodies, and the infrared fluorescence image was obtained using an Odyssey infrared imaging system (LI-CORBiosciences, Lincln, NE, USA).

The ACD-plasma of patients was collected in BD Vacutainer^® and maintained on ice during the procedures of depletion to avoid the activation of the complement. IgG depletion was obtained by passing human plasma-citrate at 10% diluted in TSB (3 mL) on a protein-G Sepharose^TM Fast Flow (GE Healthcare, Uppsala, Sweden) column, as indicated by the manufacturer’s instructions. Bound IgG were eluted with 0.1 M Glycine-HCl pH 2.8 and measured using the Pierce^TM Coomassie (Bradford reagent) protein assay kit (ThermoFisher Scientific, Waltham, MA, USA). The actual depletion of IgGs was evaluated by Western blot analysis after loading 1 µL/lane ACD-plasma on SDS-PAGE (10–12% acrylamide-bis; Bio-Rad Laboratories, Milan, Italy) and the use of horseradish peroxidase (HRP)-conjugated goat anti-human IgG (1 µg/mL; Jackson ImmunoResearch, West Grove, PA, USA).

Nicotiana benthamiana ΔXTFT plants were grown in a growth chamber at 22°C with a 16-h light/8-h dark photoperiod. All fusion proteins and antibodies or antibody fragments were transiently expressed via agro-infiltration. Total soluble proteins (TSP) and proteins secreted to the apoplastic fluid (AF) were collected 4–5 days post-infiltration (Kriechbaum et al., 2020 (link)). Expression of EPO-fusions was analyzed by immunoblotting of SDS–PAGE or native PAGE using mouse anti-EPO (1:5000, MAB2871, R&D Systems, Minneapolis, MN, United States), anti-human IgG-HRP (HC and LC, 1:10,000, W4031, Promega, Mannheim, Germany), anti-mouse IgG-HRP (1:10,000, Sigma-Aldrich, A0545), and anti-human kappa-chain-HRP (LC, 1:1000, Sigma-Aldrich, A7164) antibodies. EPO-Fc variants were purified from agroinfiltrated leaves (800 mg) with rProtein A or Protein G Sepharose^TM Fast Flow (GE Healthcare) and with KappaSelect^TM (GE Healthcare) for EPO-CL, according to the manufacturer’s instructions. Heavy chains of cetuximab (Castilho et al., 2015 (link)), anti-ebola 13F6 (Castilho et al., 2011a (link)) antibodies, and 2G12-ScFvFc (Loos et al., 2011 (link)) were cloned previously, and their expression was analyzed with anti-human gamma-chain-HRP (HC, 1:1000, Sigma-Aldrich, A7164). All purified proteins were stained with Coomassie Brilliant Blue.

For the western blotting analysis, treated cells were collected and disrupted in RIPA Lysis Buffer containing protease inhibitors (Roche Diagnostics, Berlin, Germany, 04693116001) then analyzed with polyacrylamide gel electrophoresis and the binds were visualized using an ImageQuant LAS 500(GE Healthcare, Glattbrugg, Switzerland).
For the IP analysis, treated cells were collected and disrupted in IP Lysis Buffer containing protease inhibitors, total cell extracts (1 mg per sample) were mixed with precleared protein G sepharose TM Fast Flow (GE Healthcare, 17-0618-01) and appropriate antibodies, followed by incubation for 4 h at 4 °C. The beads were collected by centrifugation, washed five times using washing buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 0.4% NP-40, and 5 mM MgCl₂), resuspended in 2 × SDS loading buffer and then subjected to western blotting as described previously.

The α-syn incubated in culture medium containing NS or PD plasma was purified by immunoprecipitation. Ten µg of 3D5 anti-α-syn antibody and 50 µL of Protein G Sepharose Fast Flow (GE, San Diego, CA, USA) were incubated with 500 µL of α-syn-containing medium at 4°C for 24 h. Next, the mixture was centrifuged at 1500 ×g for 5 min at 4°C. The pellet was washed several times with IP buffer (10 mM Tris-Cl at pH 7.5, 150 mM NaCl, 2 mM EDTA, and 0.5% Triton X-100), and purity was assessed by western blotting.
The immunoprecipitated α-syn samples were analyzed by liquid chromatography-tandem mass spectrometry (MS/MS) on a Triple TOF 5600 Plus system (Ab Sciex, Framingham, MA, USA) in 2 phases, data-dependent acquisition (DDA) followed by SWATH acquisition of the same sample, with the same gradient conditions and sample amounts. The original MS/MS data generated by DDA were analyzed with Protein Pilot v.4.5 software against the Cricetidae component of the Uniprot database. Spectral library generation and SWATH data processing were performed using Skyline v.2.5 software. A spectral library document was automatically generated prior to target data extraction.