Amicon ultra 4 10k centrifugal filter

Human ERα36 full-length cDNA fused at N-terminal with GST and tagged at C-terminal with Strep-tag II was expressed in Escherichia coli BL21 (DE3) Codon Plus-RIL. Bacteria were cultured in LB medium at an OD⁶⁰⁰ of 0.5 with 0.1 mM IPTG, and grown for 5 h at 28 °C. Bacteria were harvested by centrifugation, resuspended in Buffer Sol-ER (50 mM HEPES pH 7.9, 180 mM NaCl, 5 mM KCl, 1 mM EDTA, 5% Glycerol, 0.05% Triton, 1 mM DTT) plus 1 mM PMSF and lysed by sonication after 1 mg/mL lysozyme treatment. After centrifugation at 13 000× g for 30 min at 4 °C, the soluble extracts were applied to a StrepTrap HP-column (GE Healthcare) equilibrated with Buffer Sol-ER. The column was then washed extensively with Sol-ER and the bound protein was eluted by 2.5 mM desthiobiotin. The column purified protein was concentrated by Amicon Ultra-4 10K centrifugal filter (Merck Millipore) and dialysed in D-Tube Dialyzer Mini (Novagen) overnight at 4 °C against Sol-ER. The chromatograph was run on an AKTA Explorer system (GE Healthcare) and the purified protein was shown as a major band of GST-ERα36 by Coomassie brilliant blue staining. A lower minor band was characterized as a premature N-terminal fragment of ERα36.

Blood was drawn from each participating veteran in the morning after a night of fasting. Whole blood was collected into 10 mL vacutainer ethylenediaminetetraacetic acid (EDTA) tubes (Becton Dickinson, Franklin Lakes, NJ, USA). Plasma samples were prepared after removing blood cells by centrifugation for 10 min at 1100× g, aliquoted, and stored at −80 until subsequent use. To remove cell debris and platelets, plasma was centrifuged for 10 min at 10,000× g. EVs in plasma samples were isolated using size-exclusion chromatography as previously described [28 (link)]. In brief, 100 μL of plasma was added to the PBS equilibrated column (iZON science, Cambridge, MA, USA) and the sample was fractionated in 500 μL increments with 15 mL of PBS. Fractions 7–9 were combined as the EV fraction, and fractions 12–32 were combined as the depleted fraction. Both the EV and EV-depleted fractions were concentrated using an Amicon Ultra-4 10 K centrifugal filter (EMD Millipore, Billerica, MA, USA). Total RNA, including miRNA, was isolated from all three fractions (whole plasma, EV and EV-depleted) using a modified Qiagen miRNeasy Micro procedure (Qiagen, Germantown, MD, USA). The RNA quality and the concentration was assessed using an RNA Pico assay on an Agilent Bioanalyzer (Santa Clara, CA, USA).

Protein extraction was conducted as described previously (Wakabayashi et al., 2015 (link)) at 4 °C with minor modifications. Germinating seeds (~40 mg) of O. minor were frozen in liquid nitrogen and disrupted with a ball mill (20 Hz, 5 min; MN301, Verder Scientific, Haan, Germany). Extraction buffer (1.5 ml, pH 7.0) composed of 50 mM HEPES, 1 mM DTT, 1 mM EDTA, 20 mg polyvinylpolypyrrolidone (PVPP), and 1% Protease Inhibitor Cocktail (Merck, Kenilworth, NJ, USA) was added and incubated for 5 min. The homogenate was centrifuged at 12 000 ×g for 15 min and 0.8 ml of supernatant was collected. The residue was re-extracted in extraction buffer without PVPP, and 0.7 ml of extract was collected after centrifugation and combined with the first extract. The combined extracts (1.5 ml) were used as a soluble enzyme fraction. The enzyme extract was desalted on a PD-10 column (Cytiva, Marlborough, MA, USA) previously equilibrated with 50 mM HEPES buffer (pH 7.0) and concentrated with an Amicon Ultra-4 10K centrifugal filter (Merck).

Pelleted cultures were individually lysed using BugBuster^® Protein Extraction Reagent (EMD Millipore) with Benzonase^® Nuclease and rLysozyme™ added. Purification of each protein was individually performed using a POROS™ 20 MC metal chelate affinity (Thermo Scientific) column. Each cell-free extract was applied to the POROS™ 20 MC column equilibrated in binding buffer (20 mm Tris-HCl, pH 7.9, 500 mm NaCl, and 20 mm imidazole) at 4 °C and then washed with 10 bed volumes of binding buffer to remove unbound proteins. Each recombinant protein was next eluted using elution buffer (20 mm Tris-HCl, pH 7.9, 500 mm NaCl) containing imidazole initially at a concentration of 150 mm and then 300 mm.
Individual fractions of each recombinant proteins were subjected to SDS-PAGE using a Mini-PROTEAN^® TGX™ precast gel, 4–20% gradient (Bio-Rad), with visualization done by silver staining. Fractions containing each of the recombinant proteins (Fig. S1) were individually pooled, and the buffer was exchanged to 25 mm Tris-HCl (pH 7.9) using a PD10 column (GE Healthcare), following which the resulting protein solutions were individually concentrated using an Amicon^® Ultra-4 10K centrifugal filter (Millipore). Protein quantification was carried out using the Bradford assay (Bio-Rad) microassay procedure. Typically, 5–7 mg of each pure protein were obtained from a 30-ml E. coli culture.