Chapters authored
Clustering of Olive Pollens into Model Cultivars on the Basis of Their Allergenic Content
Part of the book: Current Insights in Pollen Allergens
Detection and Quantitation of Olive Pollen Allergen Isoforms Using 2-D Western Blotting
Part of the book: Current Insights in Pollen Allergens
Involvement of Climatic Factors in the Allergen Expression in Olive Pollen
Part of the book: Current Insights in Pollen Allergens
Pollen Allergenicity is Highly Dependent on the Plant Genetic Background: The “Variety”/“Cultivar” Issues
Part of the book: Current Insights in Pollen Allergens
A Protocol for Minimal Single Protein Labeling with CyDye Fluors for Live Cell Internalization Assays
View all chaptersIndividual 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