title: Immunoisolation of TCR signaling complexes from Jurkat T leukemic cells.
abstract: The formation of multimolecular assemblies of signaling molecules at the plasma membrane is key to triggering signaling cascades following activation of transmembrane receptors at the plasma membrane. We have developed a method to immunoisolate activated T cell receptors (TCRs) and associated signaling molecules in plasma membrane subdomains from Jurkat T leukemic cells. The immunoisolation procedure for the signaling complexes uses magnetic beads, which are coupled to TCR-activating antibodies. Following mechanical cellular disruption using nitrogen cavitation, conjugates of these beads with Jurkat cells are formed and isolated. TCR-signaling complexes within plasma membrane fragments associate with the antibody-coupled beads and are retrieved along with the beads using a magnet. We found that the immunoisolated plasma membrane fragments are highly enriched for activated TCRs and associated signaling proteins. This isolation procedure allows a detailed and precise biochemical analysis of the assembly of signaling proteins in plasma membrane subdomains.
authors.affiliation: The Basel Institute for Immunology, Grenzacherstr. 487, CH-4005, Basel, Switzerland. harder@bii.ch
authors.forename: T
authors.initials: T
authors.lastname: Harder
authors.forename: M
authors.initials: M
authors.lastname: Kuhn
mesh_terms: D000911:Antibodies, Monoclonal / Q000378:metabolism; D006801:Humans; D007158:Immunologic Techniques / Q000295:instrumentation; D019169:Jurkat Cells / Q000737:chemistry* / Q000378:metabolism*; D015458:Leukemia, T-Cell / Q000378:metabolism*; D046911:Macromolecular Substances; D008280:Magnetics; D008863:Microspheres; D011948:Receptors, Antigen, T-Cell / Q000276:immunology / Q000302:isolation & purification*; D015398:Signal Transduction
chemical_list: D000911:Antibodies, Monoclonal; D046911:Macromolecular Substances; D011948:Receptors, Antigen, T-Cell