A Protocol for Minimal Single Protein Labeling with CyDye Fluors for Live Cell Internalization Assays

Individual proteins chemically labeled with fluorescent dyes can be localized and tracked in real-time experiments in order to get insights about the site and molecular mechanism of action. Here, we have adapted a protocol that was originally developed for two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) applications, to label proteins with CyDye fluors for single-molecule internalization assays in living cells. This “minimal labeling” method offers a number of advantages including specificity and known stoichiometry, simplicity, high reproducibility, and sensitivity and allows multiplexing while minimizing perturbations of the biological system. Moreover, since only a single lysine (Lys) residue per protein molecule is labeled, this method is also quantitative. To validate experimentally our protocol, we carried out the fluorescent labeling of IBB1, a major soybean protease isoinhibitor of the Bowman-Birk family that is currently being investigated as colorectal chemopreventive agent. Then, we analyzed the in vivo internalization dynamics of the labeled IBB1 protein in human colorectal adenocarcinoma HT29 cells