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 - http://www.scopus.com/inward/record.url?scp=34347217079&partnerID=8YFLogxK
U2 - 10.1016/j.jim.2007.05.003
DO - 10.1016/j.jim.2007.05.003
M3 - Article
C2 - 17568608
AN - SCOPUS:34347217079
VL - 324
SP - 74
EP - 83
JO - Journal of immunological methods
JF - Journal of immunological methods
SN - 0022-1759
IS - 1-2
ER -