Juan de Dios Alche

By José Carlos Jiménez-López, María Isabel Rodríguez-García and Juan de Dios Alche

Part of the book: Systems and Computational Biology

By Sonia Morales, Antonio Jesús Castro, Carmen Salmerón, Francisco Manuel Marco, María Isabel Rodríguez-García and Juan de Dios Alche

Part of the book: Current Insights in Pollen Allergens

By Krzysztof Zienkiewicz, Estefanía García-Quirós, Juan de Dios Alché, María Isabel Rodríguez-García and Antonio Jesús Castro

Part of the book: Current Insights in Pollen Allergens

By Jose C. Jimenez-Lopez, Sonia Morales, Dieter Volkmann, Juan D. Alché and María I. Rodriguez-Garcia

Part of the book: Current Insights in Pollen Allergens

By Sonia Morales, Antonio Jesús Castro, María Isabel Rodríguez-García and Juan de Dios Alche

Part of the book: Current Insights in Pollen Allergens

By Juan de Dios Alché, Adoración Zafra, Jose Carlos Jiménez-López, Sonia Morales, Antonio Jesús Castro, Fernando Florido and María Isabel Rodríguez-García

Part of the book: Current Insights in Pollen Allergens

By Antonio J. Castro, Alfonso Clemente and Juan de Dios Alché

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.

Part of the book: Fluorescence Methods for Investigation of Living Cells and Microorganisms