Immunoisolation of TCR signaling complexes from Jurkat T leukemic cells.
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.
Harder
T
T
The Basel Institute for Immunology, Grenzacherstr. 487, CH-4005, Basel, Switzerland. harder@bii.ch
Kuhn
M
M
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
D000911:Antibodies, Monoclonal; D046911:Macromolecular Substances; D011948:Receptors, Antigen, T-Cell