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A native antibody-based mobility-shift technique (NAMOS-assay) to determine the stoichiometry of multiprotein complexes
- Source :
-
Journal of Immunological Methods . Jul2007, Vol. 324 Issue 1/2, p74-83. 10p. - Publication Year :
- 2007
-
Abstract
- 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. [Copyright &y& Elsevier]
- Subjects :
- *IMMUNOGLOBULINS
*STOICHIOMETRY
*PHYSICAL & theoretical chemistry
*LYMPHOCYTES
Subjects
Details
- Language :
- English
- ISSN :
- 00221759
- Volume :
- 324
- Issue :
- 1/2
- Database :
- Academic Search Index
- Journal :
- Journal of Immunological Methods
- Publication Type :
- Academic Journal
- Accession number :
- 25620670
- Full Text :
- https://doi.org/10.1016/j.jim.2007.05.003