α lactalbumin

BSA (Thermo Scientific, #J10856-22) and α-lactalbumin (Sigma, L6010) were conjugated to AF488 or AF594 (Invitrogen, #A20000 or #A20104) according to the manufacturer protocol. After proteins were conjugated, the excess free dye and DMSO were removed from the fluorescent tracer by repeated washing using centrifugal molecular weight cutoff filters (Millipore Sigma, Amicon Ultra-15, #UFC903024). The filtrate fluorescence was monitored on an inverted fluorescence microscope. The final protein concentration was measured using a BCA assay (Pierce) and the proteins were aliquoted and stored at –80°C. On the day of use, an aliquot was thawed, and the protein was added to 2 mL of Krebs buffer to a final concentration of 150 nmol mL^–1 for each fluorescent tracer to maintain similar brightness.
Other fluorescent tracers that were used included: sodium fluorescein (NaFl), 3 kDa dextran conjugated to AF488 or Texas Red (TR), and 70 kDa dextran conjugated to AF488 or TR. All fluorescent tracers were used at a final concentration of 150 nmol mL^–1.

The milk protein samples, which included κ-CN, α_S-CN, β-CN, BSA, β-lactoglobulin and α-lactalbumin, were obtained from Sigma Co. (St. Louis, MO, USA). MTGase was obtained from Ajinomoto Co., Inc. (1.0 units/mg). To investigate the MTGase-induced cross-linking of milk proteins, each milk protein sample and 2.0 units/mL MTGase were added into a 0.05 M phosphate buffer solution (pH 6.8) and dissolved. Each milk protein sample without/with 2.0 units/mL MTGase was incubated in a dry bath incubator for 0, 1, 2, or 3 h at 30 °C. Milk protein samples were heated to 80 °C for 3 min to inactivate the MTGase. Each sample was analyzed in triplicate.

RCMLA was prepared essentially as described in Ferber and Ciechanover (1986) (link). Briefly, α-lactalbumin (Sigma–Aldrich, St. Louis, MO) was denatured in 0.40 M Tris–HCl, pH 8.6, 5 mM EDTA, 6.0 M guanidine-HCl, and 50 mM DTT under N₂ gas at 37°C for 1 hr. After addition of Na-iodoacetate to a final concentration of 100 mM, the denatured protein was incubated for 1 hr. The resulting sample was dialyzed against 10 mM K-Pi, pH 7.5, and 150 mM KCl. Carboxymethylation and denaturation were confirmed by SDS-PAGE and circular dichroism spectrometry, respectively.

Casein was obtained from the bovine milk using the isoelectric precipitation generated by the College of Food, Northeast Agriculture university (Harbin, Heilongjiang Province, China). Xylose was purchased from the Huishi biochemical reagent co., LTD (Shanghai, China). ASI.398 neutral protease (from Bacillus subtilis, 50,000, U/g), was purchased from Wuxi Enzyme Preparation Company (Wuxi, Jiangsu Province, China). OPA (O-phthaldialdehyde), bovine serum albumin (66,409 Da), ovalbumin (44,300 Da), trypsin inhibitor (20,100 Da), β-lactoglobulin (36,000 Da), lysozyme (14,300 Da), α-lactalbumin (14,147 Da), oxidized glutathione (612.63 Da), reduced glutathione (307.32 Da), L-leucine, trifluoroacetic acid (TFA), potassium ferricyanide, and 2,2-diphenyl-1-picryl-hydrazyl (DPPH) were purchased from the Sigma-Aldrich Co. (St. Louis, MO, USA).

Isopropyl-β-D-thiogalactopyranoside (IPTG) was purchased from Anatrace (Maumee, OH, USA). Lysogeny broth medium was from Becton Dickinson (Franklin Lakes, NJ, USA) and terrific broth was from Formedium (Norfolk, UK). The peptides used in this study had amidated C-termini and were, with two exceptions, purchased from GL Biochem Ltd (Shanghai, China). The exceptions were the S2-P25A peptide with a selectively labeled (N¹⁵C¹³) proline residue, which was from JPT (Berlin, Germany), and suc-ALPF-pNA, which was obtained from Bachem (Bubendorf, Switzerland). The sequences of all used peptides are given in Table 1. α-Lactalbumin was from Sigma-Aldrich (St. Louis, MO, USA) and the permanently unfolded state of RCM-α-Lactalbumin was prepared by reduction and carboxymethylation, as described [56 (link)]. Crystallization reagents were from Qiagen (Germantown, MD, USA). All other chemicals were of analytical grade and obtained from Sigma-Aldrich, unless otherwise stated.

Tunicamycin, 4-phenylbutylate, cycloheximide, ibuprofen, and lysozyme were obtained from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). The ALDH2 protein (18–517 aa) was obtained from ATGen (Seoul, Korea). (±)-Flurbiprofen were from Sigma (St Louis, MO, USA) or Cayman Chemical (Ann Arbor, MI, USA). α-Lactalbumin, aspirin, and meloxicam were from Sigma. Human recombinant leptin for use in vitro was from Sigma and mouse recombinant leptin for use in vivo was from R'D Systems (Minneapolis, MN, USA). Ferriteglycidyl methacrylate (FG) beads (epoxy beads: TAS8848N1110) were from Tamagawa Seiki (Tokyo, Japan). 4′-hydroxy flurbiprofen was obtained from Toronto Research Chemicals (Toronto, ON, Canada).

The protein contents of the supernatant, nonbound, affinity pool, concentrate, and dialysate fractions, were measured by the Lowry method [12 (link)] using bovine serum albumin (BSA) as a standard. SDS PAGE was done in order to check for purity and to determine the molecular weight of the AgGSTε2. The purified protein, AgGSTε2, was subjected to polyacrylamide gel electrophoresis in the presence of 0.1% sodium dodecyl sulphate (SDS) according to the method of Laemmli [28 (link)]. SDS PAGE was done using 15% vertical slab gels and corun with a standard GST and Sigma low-range molecular weight markers, apoprotinin, α-lactalbumin, trypsin inhibitor, trypsinogen, bovine albumin, carbonic anhydrase, glyceraldehydes-3-phosphate dehydrogenase, ovalbumin, and albumin ranging from 6500 to 66000 Da (Sigma, St. Louis, MO, USA) using a BioRad Protean system (Biorad Laboratories, California, USA). The protein bands were stained using Coomassie G Stain (0.025% Coomassie G250, 40% methanol, and 7% acetic acid) overnight, destained for 1 hr in 50% methanol and 10% acetic acid, and then further destained in 5% methanol and 7% acetic acid solution. The gel picture was taken using a gel documentation station Minibis Bioimaging System (Minibis Bioimaging Systems, DNR Bioimaging System, Israel).