Ammonium sulfate

Deionized water was used in all capillary solutions for the equilibration experiments. The polymeric reservoir solutes included PEG 4000 and 8000 (Hampton Research, Aliso Viejo, CA, USA), and PEG 400 (Fluka Chemical, Milwaukee, WI, USA). The PEG reservoir solution concentrations were kept at 15%(w/v) except for PEG 400 which was 15%(v/v). All salt reservoir solutions were kept at 10%(w/v) concentration. The salt precipitants include sodium malonate and lithium sulfate (MP Biomedicals, Irvin, CA, USA), sodium chloride (Sigma–Aldrich, St Louis, MO, USA), sodium potassium tartrate, ammonium phosphate and ammonium sulfate (Fischer Scientific, Pittsburg, PA, USA).

Ammonium sulfate (Thermo Scientific, MA, USA), Gibco phosphate-buffered saline (PBS) solution 10× pH 7.4 (Thermo Scientific, MA, USA), glycerol (VWR, PA, USA), acetonitrile (ACN) (VWR, PA, USA), and deionized water (DI-H₂O) from an Elga PURELAB flex water purification system (18.2 MΩ-cm, Veolia Water Technologies, High Wycombe, UK) were used for mobile phase preparations. A silica-bead standard (QS2503 (230-260 nm), NanoFCM Nottingham, UK), was used to confirm the MALS particle count and sizing measurements. Chinese hamster ovary (CHO) cell supernatant samples obtained from the Harcum Lab (Department of Bioengineering, Clemson University) were used for all separations. Following collection from the bioreactor, the supernatant was centrifuged at 1000 × g and stored at −20 °C, before use. On the day of the separations, the supernatant was filtered with a 0.22-μm polyethersulfone (PES) filter. As a benchmark, the concentration of the IgG product in the supernatants was independently determined to be 0.71 mg mL⁻¹.

A549 cells were purchased from ATCC. The cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum at 37 °C in a humidified atmosphere at 5% CO₂. To profile the secretome, cells were cultured in serum-free RPMI for 72 h. The conditioned medium was collected, and proteins in 20 mL of the medium were precipitated with 50% ammonium sulfate (Merck, Darmstadt, Germany). The proteins were then centrifuged at 4000× g for 30 min at 4 °C, and the protein pellet was dissolved in 1 mL Tris-HCl buffer (20 mM Tris-Cl at pH 8.0). The protein solution was then transferred to dialysis tubing (ThermoFisher, Waltham, MA, USA) with a molecular weight cutoff (MWCO) of 8000–6000 Da, and dialysis was carried out to discard remnant ammonium sulfate in the protein solution. After dialysis, the protein solution was brought up to 2 mL, and secretome proteins (10 μg) were subjected to proteomic analysis.

Purification of hADPN from OVA ADPN KI chicken EW was conducted according to previously established protocols [29 (link)]. Briefly, EW was combined with five times its volume of 40% ammonium sulfate (Thermo Fisher Scientific, Waltham, MA, USA) and stirred for 4 h at 4 °C. Subsequently, the mixture was centrifuged at 3000 g for 1 h at 4 °C, and the resulting pellet was resuspended in four volumes of 20 mM Tris-HCL and 50 mM NaCl (pH 8.0) to reach the original EW volume. The sample was filtered through a 0.2 μm bottle filter and loaded onto a 5 mL HiTrap Q column (Cytiva, Marlborough, MA, USA). The protein was eluted with 20 mM Tris-HCl and 1 M NaCl (pH 8.0), and further purified via size-exclusion chromatography using a Superdex 200 Increase 10/300 GL column (Cytiva) pre-equilibrated with 20 mM Tris-HCl and 50 mM NaCl (pH 8.0).

Ethanol (99%), acetone (≥99%), sulfuric acid (96%), oxygen peroxide (50%), and Nessler’s reagent were obtained from VWR chemicals (Leuven, Belgium). Celite, sodium hydroxide (1N solution), and hydrochloric acid (≥32%) were acquired from Sigma-Aldrich (Steinheim, Germany). MES hydrate (>99%) and ammonium sulfate were provided by Alfa Aesar (Kandel, Germany). TRIS (>99.8%) was obtained from Acros Organics (Geel, Belgium). Petroleum ether (60‒80 °C) was supplied by LAB-SCAN analytical sciences (Gliwice, Poland). Amylase thermostable (3000 U/mL), protease (9 tyrosine equivalent units/mg), and amyloglucosidase (3260 U/mL), suitable for AOAC International total dietary fiber and starch analytical procedures, were obtained from Megazyme (Te Huissen, The Netherlands). Ultrapure water was prepared in a Simplicity UV water purification system (Millipore, Molsheim, France).

All consumables were of ultra-pure or electrophoresis grade. Electrophoresis apparatuses, ReadyStrip^TM IPG Strips (7 cm, pH 3–10 non-linear), Bio-Lyte^TM carrier ampholytes, and Precision Plus Protein^TM Unstained Standards (10–250 kDa) were supplied by Bio-Rad Laboratories (Hercules, CA, USA). Isolated protein standards (Bovine Serum Albumin (BSA), Chicken Egg Albumin (CEA) and Chicken Egg Lysozyme (CEL)), and 2-hydroxyethyl disulfide (HED) were supplied by Sigma-Aldrich (St. Louis, MO, USA). Acrylamide/bis-acrylamide (40%, 37.5:1) solution, components of the protease inhibitor (PI) cocktail, thiourea, urea, N,N,N’,N’-tetramethylethylenediamine, sodium n-dodecyl sulfate, ammonium sulfate, glycerol, phosphoric acid, ammonium peroxydisulfate, and tributylphosphine (TBP) were obtained from Alfa Aesar (Haverhill, MA, USA). Sodium chloride, methanol, mineral oil, and dithiothreitol (DTT) were from Thermo Fischer Scientific (Waltham, MA, USA). Acrylamide and citric acid were obtained from BioShop Canada Inc (Burlington, ON, Canada). Coomassie Brilliant Blue G-250, CHAPS, tris hydrochloride, Tris-Glycine-SDS powder, and low-melting agarose were supplied by VWR (Radnor, PA, USA). Double glass-distilled water (ddH₂O) was used throughout.