Enrichment of Colostrum Whey Proteins

Whole colostrum samples (n = 1 sample/treatment) were thawed at 4°C overnight and fractionated and enriched as described by Tacoma et al. (2016) (link). Briefly, mammalian protease inhibitor (Protease Inhibitor Cocktail, Sigma) was added to samples before centrifugation at 4,000 × g for 15 min at 4°C. The skim fraction was isolated and underwent a second centrifugation using the same conditions. The separated skim milk fraction was collected and to this fraction 60 mM of CaCl₂ was added for CN depletion as outlined by Kunz and Lonnerdal (1990) . The pH of each sample was adjusted to using 30% acetic acid (Fisher Scientific, Fair Lawn, NJ) and centrifuged at 189,000 × g for 70 min at 4°C. The supernatant was collected, frozen to −80°C, then lyophilized. One hundred milligrams of the resulting whey powder was reconstituted in PBS, and the protein concentration of each sample was determined using the bicinchoninic acid assay kit (Pierce Biotechnology, Rockford, IL). The low abundance protein fraction of each sample was enriched using the ProteoMiner kit (Bio-Rad Laboratories, Hercules, CA), where 10 mg of protein was loaded onto each column and enrichment was performed as per kit instructions. The protein concentration of each sample was determined using the bicinchoninic acid assay kit (Pierce Biotechnology).