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A native antibody-based mobility-shift technique (NAMOS-assay) to determine the stoichiometry of multiprotein complexes

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Swamy,  Mahima
Research Group and Chair of Molecular Immunology of the University of Freiburg, Max Planck Institute of Immunobiology and Epigenetics, Max Planck Society;

Minguet,  Susana
Max Planck Society;

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Siegers,  Gabrielle M.
Research Group and Chair of Molecular Immunology of the University of Freiburg, Max Planck Institute of Immunobiology and Epigenetics, Max Planck Society;

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Schamel,  Wolfgang W. A.
Research Group and Chair of Molecular Immunology of the University of Freiburg, Max Planck Institute of Immunobiology and Epigenetics, Max Planck Society;

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Citation

Swamy, M., Minguet, S., Siegers, G. M., Alarcón, B., & Schamel, W. W. A. (2007). A native antibody-based mobility-shift technique (NAMOS-assay) to determine the stoichiometry of multiprotein complexes. Journal of Immunological Methods, 324, 74-83.


Cite as: http://hdl.handle.net/11858/00-001M-0000-002B-91F6-8
Abstract
Characterization of multiprotein complexes (MPCs) is an important step toward an integrative view of protein interaction networks and prerequisite for a molecular understanding of how a certain MPC functions. Here, we present a technique utilizing monoclonal subunit-specific antibodies for an electrophoretic immunoshift assay in Blue Native-gels (NAMOS-assay), which allows the determination of the stoichiometry of MPCs. First, we use the B cell antigen receptor as a model MPC whose stoichiometry is known, confirming the HC2LC2Igα/β1 stoichiometry. Second, we demonstrate that the digitonin-extracted T cell antigen receptor (TCR) extracted from T cells has a stoichiometry of αβε2γδζ2. We then show that the NAMOS-assay does not require purified MPCs, since it can determine the stoichiometry of an MPC in cell lysates. The NAMOS-assay is also compatible with use of epitope tags appended to the protein of interest, as e.g. the widely used HA-tag, and anti-epitope antibodies for the assay. Given its general applicability, this method has a wide potential for MPC research.