To characterize the structural effects of catechol-mediated reactions on BSA, 1.5-mL reaction mixtures were prepared in microtubes, with fixed catechol and Pi buffer concentrations of 15.6 mM and 50 mM (pH 7.8), respectively. BSA concentrations were set to 2 mg/mL, 16 mg/mL, or 32 mg/mL. Mixtures were pre-warmed to 37 °C, and reactions were initiated by adding 150 L of 156 mM catechol solution. Reactions were maintained at 37 °C in a mini dry bath. At time points 0 h, 0.5 h, 1.5 h, 2.0 h, 2.5 h, 3.0 h, 4.0 h, 5.0 h, and 6.0 h, 100 L samples were withdrawn for analysis. Preparations containing 2 mg/mL BSA were used undiluted, while preparations with 16 or 32 mg/mL BSA were diluted to 2 mg/mL with deionized water before mixing with sample buffer and boiling. 50 L of the 2 mg/mL BSA samples -as well as the 16 mg/mL and 32 mg/mL preparations, now also normalized to 2 mg/mL -were mixed with 250 L of 6x SDS-PAGE sample loading buffer (375 mM Tris-HCl (pH 6.8), 9% (v/v) SDS, 50% (v/v) glycerol, 9% (v/v) BME, 0.03% (w/v) BPB), boiled for 5 min on a mini dry bath, then immediately cold-snapped on ice and stored at -20 °C until use. Samples were subjected to SDS-PAGE using 8% polyacrylamide gels hand-cast with ProtoGel 30% acrylamide solution, 4x resolving buffer, and stacking buffer, using Bio-Rad Mini-PROTEAN ® short glass plates and 1 mm glass spacer plates. The gels were run in a Bio-Rad Mini-PROTEAN Tetra Cell system using 1x Tris/glycine buffer (diluted from 10x buffer). 4 g of sample protein (12 L) were loaded per well along with 3 L of PageRuler ™ Plus prestained protein ladder. Electrophoresis was carried out at a constant voltage of 200 V until the BPB dye front reached the bottom of the gel. After electrophoresis, gels were stained in CBB solution (0.1% (w/v) CBB, 40% (v/v) methanol, 10% (v/v) acetic acid) until darkly opaque, then destained with 50% methanol and 10% acetic acid using knotted laboratory wipes to absorb liberated colloidal stain, until strong contrast was achieved between background and protein bands. Gels were transilluminated with a white light box and digitally imaged using a Google Pixel 8.