D glucitol

Cellobiose, D-glucos, D-xylose, methyl-α-D-glucoside, methyl-β-D-glucoside, D-mannitol, D-glucitol, N-acetyl-D-glucosamine, D-glucose-1-phosphate, L-arabinose, D-frutose, D-galactose, lactose, laminaribiose, and glucose 1-phosphate (Glu-1P) were purchased from Sigma-Aldrich (USA).

The extracted nail proteins were transferred into a 2 mL boronate affinity column [Glass Econo-Column (Bio-Rad Laboratories Inc., Richmond, CA, USA)] to undergo a chromatographic separation into glycated nail proteins and nonglycated nail proteins. The wash buffer consisted of 0.25 mol/L ammonium acetate, containing 0.05 mol/L magnesium chloride, pH: 8.3. The elution buffer consisted of 0.1 mol/L Tris-HCl, containing 0.2 mol/L sorbitol (D-Glucitol, Sigma Aldrich, St. Louis, MO, USA) and 0.05 mol/L EDTA [34 (link)]. The concentration of proteins was assayed by the pyrogallol red-molybdate method on a Cobas 8000 analyzer [35 (link)]. The percentage of the glycated protein fraction was calculated from the protein content of both fractions. Following chromatography, binding and nonbinding fractions were analyzed using electrophoresis.