Bt pe maleimide

Biotin-labeled proteins were prepared by incubating 5 mg/ml BSA (Iwai Chemicals), human serum albumin (FUJIFILM Wako Pure Chemical, Inc), transferrin (FUJIFILM Wako Pure Chemical, Inc), and IgG (FUJIFILM Wako Pure Chemical, Inc) with a 10-fold molar Bt-PE-maleimide (Dojindo Laboratories) in PBS (10 mM sodium phosphate buffer (pH 7.4)) at 25 °C for 16 h. After incubation, the aliquots were dialyzed against PBS. The polyphenol-modified proteins were prepared by incubating 1 mg/ml proteins or biotinylated proteins with 2.5 mM polyphenols in PBS containing 10% dimethyl sulfoxide for 72 h at 37 °C under atmospheric oxygen. After incubation, the reactants were collected and dialyzed with PBS. The protein concentrations were measured by a bicinchoninic acid assay (Nacalai Tesque, Inc). Bt-AET was synthesized according to previous studies (28 ). The polyphenol-modified amino acid analogs were prepared upon incubation of 1 mM Bt-APA (Thermo Fisher Scientific, Inc) or Bt-AET with 2.5 mM polyphenols in PBS containing 10% dimethyl sulfoxide for 24 h at 37 °C under atmospheric oxygen. The oxidized Bt-APA was prepared according to a previous report (2 (link)).

Bt-labeled BSA (Bt-BSA) was prepared by incubating 5 mg/ml BSA (Iwai Chemicals) with a 10-fold molar Bt-PE-maleimide (Dojindo Laboratories) in PBS at 25 °C for 16 h. After incubation, the aliquots were dialyzed against PBS. Biotin-labeled Plg (Bt-Plg) was prepared with EZ-Link Sulfo-NHS-LC-Biotin (Thermo Fisher Scientific) following the manufacturer’s instructions. The AGEs were prepared according to a previous report^{9 (link)} by incubating 1.0 mg/ml BSA with 25 mM DHA (Sigma) in PBS at 37 °C under atmospheric oxygen. After 72 h, the reactants were collected and dialyzed with PBS. For the preparation of Bt-labeled acylated proteins (Ac-BSA, Sc-BSA, and Ma-BSA), 5 mg/ml Bt-BSA was added to equal volume of a saturated solution of sodium acetate with continuous stirring on ice, followed by the incubation at 4 °C for 1 h with 2 mM acetic anhydride, succinic anhydride, or maleic anhydride, respectively. After incubation, the aliquots were dialyzed against PBS. The protein concentrations were measured by a BCA assay (Nacalai Tesque Inc.). DHA-modified Bt-labeled N-pentylamine (DHA-Bt-PA) was prepared upon incubation of 6 mM EZ-Link pentylamine biotin (Thermo Fisher Scientific) with 25 mM DHA at 37 °C for 72 h.