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

Mahima Swamy, Susana Minguet, Gabrielle M. Siegers, Balbino Alarcón, Wolfgang W.A. Schamel (Lead / Corresponding author)

Research output: Contribution to journalArticlepeer-review

27 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)74-83
Number of pages10
JournalJournal of immunological methods
Volume324
Issue number1-2
Early online date4 Jun 2007
DOIs
Publication statusPublished - 31 Jul 2007

Keywords

  • Blue Native
  • BN-PAGE
  • Composition
  • Proteomics
  • Stoichiometry
  • TCR

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