Imperial protein stain

Human plasma containing EDTA from a single healthy individual was obtained from Zen-Bio. One recombinant HRG variant, containing the mutations Pro204 and Asn493, was produced in CHO cell lines by PeproTech. Four other HRG variants, with pairwise combinations of targeted mutations for positions 204 and 493, were produced in HEK293E-253 cells (HEK293) by ImmunoPrecise. HRGs were expressed with sequences matching allelic variants on position 204 or 493 in human population: Pro204/Asn493, Pro204/Ile493, Ser204/Ile493, and Ser204/Asn493.
Cobalt-loaded resin was acquired from Thermo Scientific. Imidazole was purchased from Sigma–Aldrich. Tris–HCl, Tris(2-carboxyethyl)phosphine, chloroacetamide, sodium deoxycholate (SDC), and TFA were purchased from Sigma–Aldrich. Oasis HLB 96-well μElution Plates were purchased from Waters. Sequencing-grade trypsin was obtained from Promega. SialEXO and OpeRATOR were obtained from Genovis. Plasmin was purchased from Abcam. Formic acid (FA) and acetonitrile were acquired from Biosolve Chimie. The SDS-PAGE standards and Imperial Protein Stain were purchased from Bio-Rad and Thermo Scientific, respectively.

Yellow field pea flour (~20% protein content) was provided by AGT Foods (Regina, SK, Canada) and commercial pea protein isolate (cPPI, 81.2% protein, 3.86% ash), PURIS™ Pea Protein, was provided by Puris Foods (Minneapolis, MN, USA). Commercial whey protein isolate (cWPI, 94.6% protein, 4.10% ash), BiPro^®, was provided by Agropur Ingredients (Eden Prairie, MN, USA). Defatted soy flour (~53% protein) and commercial soy protein isolate (cSPI, ~90.7% protein), ProFam^® 974, were provided by Archer Daniels Midland (ADM) (Decatur, IL, USA). All aforementioned samples were stored at −20 °C prior to usage. SnakeSkin™ dialysis tubing (3.5 kDa cut off) and Sudan Red 7B were purchased from Thermo Fisher Scientific™ (Waltham, MA, USA). Criterion™ TGX™ 4–20% precast gels, Laemmli 4X loading buffer, Imperial™ Protein Stain, 10X Tris/Glycine/SDS running buffer and Precision Plus molecular weight marker were purchased from Bio-Rad Laboratories, Inc. (Hercules, CA, USA). Vivaflow^® membrane ultrafiltration crossflow cassettes (3 kDa MWCO) were purchased from Sartorius™ (Gottingen, Germany). All other chemical reagents and supplies were purchased from Thermo Fisher Scientific and Sigma-Aldrich.