title: An Optimized Procedure for the Site-Directed Labeling of NGF and proNGF for Imaging Purposes.
abstract: Neurotrophins are growth factors of fundamental importance for the development, survival and maintenance of different neuronal and non-neuronal populations. Over the years, the use of labeled neurotrophins has helped in the study of their biological functions, leading to a better understanding of the processes that regulate their transport, traffic, and signaling. However, the diverse and heterogeneous neurotrophin labeling strategies adopted so far have often led to poorly reproducible protocols and sometimes conflicting conclusions. Here we present a robust, reliable, and fast method to obtain homogeneous preparations of fluorescent proNGF and NGF with 1:1 labeling stoichiometry. This strategy is well suited for several applications, ranging from advanced imaging techniques such assingle particle tracking, to analyses that require large amounts of neurotrophins such asin vivomonitoring of protein biodistribution. As a proof of the quality of the labeled NGF and proNGF preparations, we provide a quantitative analysis of their colocalization with proteins involved in thesignaling endosomefunction and sorting. This new analysis allowed demonstrating that proNGF localizes at a sub-population of endosomes not completely overlapped to the one hosting NGF.
authors.affiliation: BioSNS Laboratory, Scuola Normale Superiore and Istituto di Neuroscienze - CNR Pisa, Italy.
authors.forename: Pierluigi
authors.initials: P
authors.lastname: Di Matteo
authors.affiliation: BioSNS Laboratory, Scuola Normale Superiore and Istituto di Neuroscienze - CNR Pisa, Italy.
authors.forename: Mariantonietta
authors.initials: M
authors.lastname: Calvello
authors.affiliation: NEST Laboratory, Scuola Normale Superiore and Istituto Nanoscienze - CNR Pisa, Italy.
authors.forename: Stefano
authors.initials: S
authors.lastname: Luin
authors.affiliation: BioSNS Laboratory, Scuola Normale Superiore and Istituto di Neuroscienze - CNRPisa, Italy; Center for Nanotechnology Innovation@NEST, Istituto Italiano di TecnologiaPisa, Italy.
authors.forename: Laura
authors.initials: L
authors.lastname: Marchetti
authors.affiliation: BioSNS Laboratory, Scuola Normale Superiore and Istituto di Neuroscienze - CNR Pisa, Italy.
authors.forename: Antonino
authors.initials: A
authors.lastname: Cattaneo
keywords: fluorescent NGF; fluorescent proNGF; neurotrophins labeling; neurotrophins purification; signaling endosome