title: In Situ Proximity Ligation Assay (PLA) Analysis of Protein Complexes Formed Between Golgi-Resident, Glycosylation-Related Transporters and Transferases in Adherent Mammalian Cell Cultures.
abstract: In situ proximity ligation assay (PLA) is a novel, revolutionary technique that can be employed to visualize protein complexes in fixed cells and tissues. This approach enables demonstration of close (i.e., up to 40 nm) proximity between any two proteins of interest that can be detected using a pair of specific antibodies that have been raised in distinct species. Primary antibodies bound to the target proteins are subsequently recognized by two PLA probes, i.e., secondary antibodies conjugated with oligonucleotides that anneal when brought into close proximity in the presence of two connector oligonucleotides and a DNA ligase forming a circular DNA molecule. In the next step, the resulting circular DNA is amplified by a rolling circle polymerase. Finally, fluorescent oligonucleotide probes hybridize to complementary fragments of the amplified DNA molecule, forming a typical, spot-like pattern of PLA signal that reflects subcellular localization of protein complexes. Here we describe the use of in situ PLA in adherent cultures of mammalian cells in order to visualize interactions between Golgi-resident, functionally related glycosyltransferases and nucleotide sugar transporters relevant to N-glycan biosynthesis.
authors.affiliation: Laboratory of Biochemistry, Faculty of Biotechnology, University of Wroclaw, Joliot-Curie 14A, 50-383, Wrocław, Poland.
authors.forename: Dorota
authors.initials: D
authors.lastname: Maszczak-Seneczko
authors.affiliation: Laboratory of Biochemistry, Faculty of Biotechnology, University of Wroclaw, Joliot-Curie 14A, 50-383, Wrocław, Poland.
authors.forename: Paulina
authors.initials: P
authors.lastname: Sosicka
authors.affiliation: Laboratory of Biochemistry, Faculty of Biotechnology, University of Wroclaw, Joliot-Curie 14A, 50-383, Wrocław, Poland.
authors.forename: Teresa
authors.initials: T
authors.lastname: Olczak
authors.affiliation: Laboratory of Biochemistry, Faculty of Biotechnology, University of Wroclaw, Joliot-Curie 14A, 50-383, Wrocław, Poland. Mariusz.Olczak@uwr.edu.pl.
authors.forename: Mariusz
authors.initials: M
authors.lastname: Olczak
mesh_terms: D000818:Animals; D000906:Antibodies / Q000737:chemistry*; D019556:COS Cells; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism; D002522:Chlorocebus aethiops; D011088:DNA Ligases / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry*; D005964:Glucosyltransferases / Q000737:chemistry* / Q000378:metabolism; D006056:Golgi Apparatus / Q000737:chemistry* / Q000378:metabolism; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D046912:Multiprotein Complexes / Q000737:chemistry / Q000378:metabolism; D009841:Oligonucleotides / Q000737:chemistry*
chemical_list: D000906:Antibodies; D002352:Carrier Proteins; D005456:Fluorescent Dyes; D046912:Multiprotein Complexes; D009841:Oligonucleotides; D005964:Glucosyltransferases; D011088:DNA Ligases
keywords: DNA amplification; Fluorescence detection; Glycosyltransferases; Golgi membrane; In situ proximity ligation assay (PLA); Nucleotide sugar transporters; Oligonucleotide annealing; Primary antibodies