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

TY - JOUR

T1 - A native antibody-based mobility-shift technique (NAMOS-assay) to determine the stoichiometry of multiprotein complexes

AU - Swamy, Mahima

AU - Minguet, Susana

AU - Siegers, Gabrielle M.

AU - Alarcón, Balbino

AU - Schamel, Wolfgang W.A.

PY - 2007/7/31

Y1 - 2007/7/31

N2 - 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.

AB - 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.

KW - Blue Native

KW - BN-PAGE

KW - Composition

KW - Proteomics

KW - Stoichiometry

KW - TCR

UR - https://www.scopus.com/pages/publications/34347217079

U2 - 10.1016/j.jim.2007.05.003

DO - 10.1016/j.jim.2007.05.003

M3 - Article

C2 - 17568608

AN - SCOPUS:34347217079

SN - 0022-1759

VL - 324

SP - 74

EP - 83

JO - Journal of immunological methods

JF - Journal of immunological methods

IS - 1-2

ER -