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Fluorescent labelling of BSA, AGE-2, and AGE-3 using Alexa Fluor 488 C5 maleimide

SH Shinichi Hamasaki
TK Takuro Kobori
YY Yui Yamazaki
AK Atsuhiro Kitaura
AN Atsuko Niwa
TN Takashi Nishinaka
MN Masahiro Nishibori
SM Shuji Mori
SN Shinichi Nakao
HT Hideo Takahashi
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BSA, AGE-2, and AGE-3 were fluorescently labelled as described previously with some modification15,16. Briefly, each protein was incubated with 20 times the amount of Alexa Fluor 488 C5 maleimide (Thermo Fisher Scientific) at room temperature for 2 h in phosphate buffered saline (PBS) and then dialysed with PBS at 4 °C for 2 days. Total protein concentration was quantified by the Bradford method17 using a Bradford protein assay kit (Bio-Rad Laboratories, Kidlington, UK). Alexa Fluor 488-labelled compound fluorescence intensity was measured using ARVO MX 1420 (PerkinElmer Japan, Yokohama, Japan) (excitation: 485 nm, emission: 535 nm). The strengths of Alexa Fluor 488-BSA, -AGE-2, or -AGE-3 per unit dosage were each adjusted by adding respective unlabelled proteins.

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