Detection of Protein-Protein Interactions and Posttranslational Modifications Using the Proximity Ligation Assay: Application to the Study of the SUMO Pathway.
The detection of protein-protein interactions by imaging techniques often requires the overexpression of the proteins of interest tagged with fluorescent molecules, which can affect their biological properties and, subsequently, flaw experiment interpretations. The recent development of the proximity ligation assays (PLA) technology allows easy visualization of endogenous protein-protein interactions at the single molecule level. PLA relies on the use of combinations of antibodies coupled to complementary oligonucleotides that are amplified and revealed with a fluorescent probe, each spot representing a single protein-protein interaction. Another application of this technique is the detection of proteins posttranslational modifications to monitor their localization and dynamics in situ. Here, we describe the use of PLA to detect protein SUMOylation, a posttranslational modification related to ubiquitination, as well as interaction of SUMOylated substrates with other proteins, using both adherent and suspension cells.
Equipe Labellisée par la Ligue contre le Cancer, Institut de Génétique Moléculaire de Montpellier, CNRS UMR 5535, Université de Montpellier, CNRS, 1919 Route de Mende, Montpellier, Cedex 5, 34293, France.
Marko
M
Ristic
Equipe Labellisée par la Ligue contre le Cancer, Institut de Génétique Moléculaire de Montpellier, CNRS UMR 5535, Université de Montpellier, CNRS, 1919 Route de Mende, Montpellier, Cedex 5, 34293, France.
Frédérique
F
Brockly
Equipe Labellisée par la Ligue contre le Cancer, Institut de Génétique Moléculaire de Montpellier, CNRS UMR 5535, Université de Montpellier, CNRS, 1919 Route de Mende, Montpellier, Cedex 5, 34293, France.
Marc
M
Piechaczyk
Equipe Labellisée par la Ligue contre le Cancer, Institut de Génétique Moléculaire de Montpellier, CNRS UMR 5535, Université de Montpellier, CNRS, 1919 Route de Mende, Montpellier, Cedex 5, 34293, France. guillaume.bossis@igmm.cnrs.fr.
Guillaume
G
Bossis
D000818:Animals; D001681:Biological Assay / Q000379:methods; D006801:Humans; D011485:Protein Binding / Q000235:genetics / Q000502:physiology; D025941:Protein Interaction Mapping / Q000379:methods*; D011499:Protein Processing, Post-Translational / Q000235:genetics / Q000502:physiology; D058207:Sumoylation / Q000235:genetics / Q000502:physiology
Protein–protein interaction; Proximity ligation assay; SUMOylation