NDLI: Detection of in situ protein-protein complexes at the Drosophila larval neuromuscular junction using proximity ligation assay.

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Detection of in situ protein-protein complexes at the Drosophila larval neuromuscular junction using proximity ligation assay.

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Detection of in situ protein-protein complexes at the Drosophila larval neuromuscular junction using proximity ligation assay.

Content Provider	World Health Organization (WHO)-Global Index Medicus
Author	Wang, Simon Yoo, SooHyun Kim, Hae-Yoon Wang, Mannan Zheng, Clare Parkhouse, Wade Krieger, Charles Harden, Nicholas
Description	Author Affiliation: Wang S ( Department of Molecular Biology and Biochemistry, Simon Fraser University.); Yoo S ( Department of Molecular Biology and Biochemistry, Simon Fraser University.); Kim HY ( Department of Molecular Biology and Biochemistry, Simon Fraser University.); Wang M ( Department of Biomedical Physiology and Kinesiology, Simon Fraser University.); Zheng C ( Department of Biomedical Physiology and Kinesiology, Simon Fraser University.); Parkhouse W ( Department of Biomedical Physiology and Kinesiology, Simon Fraser University.); Krieger C ( Department of Biomedical Physiology and Kinesiology, Simon Fraser University.); Harden N ( Department of Molecular Biology and Biochemistry, Simon Fraser University)
Abstract	Discs large (Dlg) is a conserved member of the membrane-associated guanylate kinase family, and serves as a major scaffolding protein at the larval neuromuscular junction (NMJ) in Drosophila. Previous studies have shown that the postsynaptic distribution of Dlg at the larval NMJ overlaps with that of Hu-li tai shao (Hts), a homologue to the mammalian adducins. In addition, Dlg and Hts are observed to form a complex with each other based on co-immunoprecipitation experiments involving whole adult fly lysates. Due to the nature of these experiments, however, it was unknown whether this complex exists specifically at the NMJ during larval development. Proximity Ligation Assay (PLA) is a recently developed technique used mostly in cell and tissue culture that can detect protein-protein interactions in situ. In this assay, samples are incubated with primary antibodies against the two proteins of interest using standard immunohistochemical procedures. The primary antibodies are then detected with a specially designed pair of oligonucleotide-conjugated secondary antibodies, termed PLA probes, which can be used to generate a signal only when the two probes have bound in close proximity to each other. Thus, proteins that are in a complex can be visualized. Here, it is demonstrated how PLA can be used to detect in situ protein-protein interactions at the Drosophila larval NMJ. The technique is performed on larval body wall muscle preparations to show that a complex between Dlg and Hts does indeed exist at the postsynaptic region of NMJs.
File Format	HTM / HTML
e-ISSN	1940087X
Journal	Journal of Visualized Experiments
Issue Number	95
Language	English
Publisher	MyJove Corp.
Publisher Date	2015-01-20
Publisher Place	United States
Access Restriction	Open
Subject Keyword	Discipline Physical Sciences Discipline Life Sciences Discipline Medicine Calmodulin-binding Proteins Metabolism Drosophila Proteins Drosophila Neuromuscular Junction Tumor Suppressor Proteins Animals Growth & Development Guanylate Kinase Immunohistochemistry Larva Chemistry Protein Interaction Mapping Research Support, Non-u.s. Gov't Video-audio Media
Content Type	Text
Resource Type	Article

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