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Sci. STKE, 27 February 2001
Vol. 2001, Issue 71, p. pl1
[DOI: 10.1126/scisignal.712001pl1]

PROTOCOLS

Immunoisolation of TCR Signaling Complexes from Jurkat T Leukemic Cells

Thomas Harder* and Marina Kuhn

The Basel Institute for Immunology, Grenzacherstr. 487, CH-4005, Basel, Switzerland.

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.

*Corresponding author, E-mail: harder{at}bii.ch.

Citation: T. Harder, M. Kuhn, Immunoisolation of TCR Signaling Complexes from Jurkat T Leukemic Cells. Sci. STKE 2001, pl1 (2001).

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