title: Labeling of platelet-derived growth factor by reversible biotinylation to visualize its endocytosis by microscopy.
abstract: Microscopical analyses of endocytic trafficking require tools for efficient detection of internalized cargo. Due to the lack of suitable reagents and limitations related to its biological properties, visualization of platelet-derived growth factor (PDGF) by microscopy remained a challenge. To overcome these restrictions, we generated a biologically active PDGF labeled with up to five biotins on cleavable linkers. Subsequently, we stimulated cells with such ligand followed by removal of extracellular biotins. PDGF captured in endocytic vesicles was successfully detected with antibiotin antibodies with parallel detection of PDGF receptor, as well as other markers of endocytic compartments. Labeled PDGF was successfully validated and can be utilized in various microscopical techniques.
authors.affiliation: Laboratory of Cell Biology, International Institute of Molecular and Cell Biology, Warsaw, Poland.
authors.forename: Łukasz
authors.initials: Ł
authors.lastname: Sadowski
authors.affiliation: Laboratory of Cell Biology, International Institute of Molecular and Cell Biology, Warsaw, Poland.
authors.forename: Kamil
authors.initials: K
authors.lastname: Jastrzębski
authors.affiliation: Laboratory of Bioinformatics and Protein Engineering, International Institute of Molecular and Cell Biology, Warsaw, Poland.
authors.forename: Elżbieta
authors.initials: E
authors.lastname: Purta
authors.affiliation: University of Birmingham, School of Biosciences, Birmingham, United Kingdom.
authors.forename: Carina
authors.initials: C
authors.lastname: Hellberg
authors.affiliation: Laboratory of Cell Biology, International Institute of Molecular and Cell Biology, Warsaw, Poland. Electronic address: miaczynska@iimcb.gov.pl.
authors.forename: Marta
authors.initials: M
authors.lastname: Miaczynska
mesh_terms: D019921:Biotinylation; D002460:Cell Line; D004705:Endocytosis; D006801:Humans; D008856:Microscopy, Fluorescence; D010982:Platelet-Derived Growth Factor / Q000737:chemistry / Q000378:metabolism*; D017479:Receptors, Platelet-Derived Growth Factor / Q000378:metabolism; D013194:Staining and Labeling
chemical_list: D010982:Platelet-Derived Growth Factor; D017479:Receptors, Platelet-Derived Growth Factor
keywords: Endocytosis; Labeling; Microscopy; PDGF; Reversible biotinylation