title: Purification of LAT-Containing Membranes from Resting and Activated T Lymphocytes.
abstract: In T lymphocytes, the immune synapse is an active zone of vesicular traffic. Directional transport of vesicular receptors and signaling molecules from or to the immune synapse has been shown to play an important role in T-cell receptor (TCR) signal transduction. However, how vesicular trafficking is regulating the activation of T cells is still a burning question, and the characterization of these intracellular compartments remains the first step to understand this process. We describe herein a protocol, which combines a separation of membranes on flotation gradient with an affinity purification of Strep-tagged fusion transmembrane proteins with Strep-Tactin®resin, allowing the purification of membranes containing the Strep-tagged molecule of interest. By keeping the membranes intact, this protocol leads to the purification of molecules physically associated with the Strep-tagged protein as well as of molecules present in the same membrane compartment: transmembrane proteins, proteins strongly associated with the membranes, and luminal proteins. The example shown herein is the purification of membrane compartment prepared from T lymphocytes expressing LAT fused to a Strep-tag.
authors.affiliation: Section Recherche, Institut Curie, INSERM U932, 26 Rue d'Ulm, 75248, Paris, Cedex 05, France.|PSL Research University, Paris, France.|Immunity and Cancer, INSERM U932, Paris, France.
authors.forename: Claire
authors.initials: C
authors.lastname: Hivroz
authors.affiliation: University of Milan, Milan, Italy.|Istituto Nazionale Genetica Molecolare 'Romeo ed Enrica Invernizzi', INGM, Milan, Italy.
authors.forename: Paola
authors.initials: P
authors.lastname: Larghi
authors.affiliation: Section Recherche, Institut Curie, INSERM U932, 26 Rue d'Ulm, 75248, Paris, Cedex 05, France.|PSL Research University, Paris, France.|Immunity and Cancer, INSERM U932, Paris, France.
authors.forename: Mabel
authors.initials: M
authors.lastname: Jouve
authors.affiliation: Section Recherche, Institut Curie, INSERM U932, 26 Rue d'Ulm, 75248, Paris, Cedex 05, France. laurence.bataille@curie.fr.|PSL Research University, Paris, France. laurence.bataille@curie.fr.|Immunity and Cancer, INSERM U932, Paris, France. laurence.bataille@curie.fr.
authors.forename: Laurence
authors.initials: L
authors.lastname: Ardouin
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry* / Q000276:immunology / Q000302:isolation & purification*; D002462:Cell Membrane / Q000737:chemistry* / Q000276:immunology; D002846:Chromatography, Affinity / Q000379:methods*; D006801:Humans; D019169:Jurkat Cells; D008213:Lymphocyte Activation; D008565:Membrane Proteins / Q000737:chemistry* / Q000276:immunology / Q000302:isolation & purification*; D011948:Receptors, Antigen, T-Cell / Q000737:chemistry / Q000276:immunology; D013601:T-Lymphocytes / Q000737:chemistry* / Q000276:immunology
chemical_list: D048868:Adaptor Proteins, Signal Transducing; C110810:LAT protein, human; D008565:Membrane Proteins; D011948:Receptors, Antigen, T-Cell
keywords: Flotation gradient; Immune synapse; LAT; Strep-tag-Strep-tactin affinity purification; T Lymphocyte