A native antibody-based mobility-shift technique (NAMOS-assay) to determine the stoichiometry of multiprotein complexes.
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 HC(2)LC(2)Igalpha/beta(1) stoichiometry. Second, we demonstrate that the digitonin-extracted T cell antigen receptor (TCR) extracted from T cells has a stoichiometry of alphabetaepsilon(2)gammadeltazeta(2). 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.
Swamy
Mahima
M
Max Planck-Institut für Immunbiologie and Universität Freiburg, Biologie III, Stübeweg 51, 79108 Freiburg, Germany.
Minguet
Susana
S
Siegers
Gabrielle M
GM
Alarcón
Balbino
B
Schamel
Wolfgang W A
WW
D000818:Animals; D000906:Antibodies / Q000378:metabolism*; D002460:Cell Line; D045744:Cell Line, Tumor; D004591:Electrophoresis, Polyacrylamide Gel; D024202:Electrophoretic Mobility Shift Assay; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000378:metabolism; D007147:Immunoglobulin Light Chains / Q000378:metabolism; D051379:Mice; D046912:Multiprotein Complexes / Q000737:chemistry* / Q000276:immunology* / Q000378:metabolism; D011947:Receptors, Antigen, B-Cell / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D011948:Receptors, Antigen, T-Cell / Q000737:chemistry / Q000378:metabolism
D000906:Antibodies; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D046912:Multiprotein Complexes; D011947:Receptors, Antigen, B-Cell; D011948:Receptors, Antigen, T-Cell