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 electr...
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| Veröffentlicht in: | Journal of immunological methods 2007-07, Vol.324 (1), p.74-83 |
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| container_title | Journal of immunological methods |
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| creator | Swamy, Mahima Minguet, Susana Siegers, Gabrielle M. Alarcón, Balbino Schamel, Wolfgang W.A. |
| description | 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
2LC
2Igα/β
1 stoichiometry. Second, we demonstrate that the digitonin-extracted T cell antigen receptor (TCR) extracted from T cells has a stoichiometry of αβε
2γδζ
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. |
| doi_str_mv | 10.1016/j.jim.2007.05.003 |
| format | Article |
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2LC
2Igα/β
1 stoichiometry. Second, we demonstrate that the digitonin-extracted T cell antigen receptor (TCR) extracted from T cells has a stoichiometry of αβε
2γδζ
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.</description><identifier>ISSN: 0022-1759</identifier><identifier>EISSN: 1872-7905</identifier><identifier>DOI: 10.1016/j.jim.2007.05.003</identifier><identifier>PMID: 17568608</identifier><identifier>CODEN: JIMMBG</identifier><language>eng</language><publisher>Amsterdam: Elsevier B.V</publisher><subject>Animals ; Antibodies - metabolism ; Biological and medical sciences ; Blue Native ; BN-PAGE ; Cell Line ; Cell Line, Tumor ; Composition ; Electrophoresis, Polyacrylamide Gel ; Electrophoretic Mobility Shift Assay ; Fundamental and applied biological sciences. Psychology ; Fundamental immunology ; Humans ; Immunoglobulin Heavy Chains - metabolism ; Immunoglobulin Light Chains - metabolism ; Mice ; Molecular immunology ; Multiprotein Complexes - chemistry ; Multiprotein Complexes - immunology ; Multiprotein Complexes - metabolism ; Proteomics ; Receptors, Antigen, B-Cell - chemistry ; Receptors, Antigen, B-Cell - immunology ; Receptors, Antigen, B-Cell - metabolism ; Receptors, Antigen, T-Cell - chemistry ; Receptors, Antigen, T-Cell - metabolism ; Stoichiometry ; TCR ; Techniques</subject><ispartof>Journal of immunological methods, 2007-07, Vol.324 (1), p.74-83</ispartof><rights>2007 Elsevier B.V.</rights><rights>2007 INIST-CNRS</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c478t-29c0599bfa0220f0dc46e471b7913638ab21e6239a4503600512c3fc51490a0b3</citedby><cites>FETCH-LOGICAL-c478t-29c0599bfa0220f0dc46e471b7913638ab21e6239a4503600512c3fc51490a0b3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktohtml>$$Uhttps://dx.doi.org/10.1016/j.jim.2007.05.003$$EHTML$$P50$$Gelsevier$$H</linktohtml><link.rule.ids>314,780,784,3541,27915,27916,45986</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=18902900$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/17568608$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Swamy, Mahima</creatorcontrib><creatorcontrib>Minguet, Susana</creatorcontrib><creatorcontrib>Siegers, Gabrielle M.</creatorcontrib><creatorcontrib>Alarcón, Balbino</creatorcontrib><creatorcontrib>Schamel, Wolfgang W.A.</creatorcontrib><title>A native antibody-based mobility-shift technique (NAMOS-assay) to determine the stoichiometry of multiprotein complexes</title><title>Journal of immunological methods</title><addtitle>J Immunol Methods</addtitle><description>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
2LC
2Igα/β
1 stoichiometry. Second, we demonstrate that the digitonin-extracted T cell antigen receptor (TCR) extracted from T cells has a stoichiometry of αβε
2γδζ
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.</description><subject>Animals</subject><subject>Antibodies - metabolism</subject><subject>Biological and medical sciences</subject><subject>Blue Native</subject><subject>BN-PAGE</subject><subject>Cell Line</subject><subject>Cell Line, Tumor</subject><subject>Composition</subject><subject>Electrophoresis, Polyacrylamide Gel</subject><subject>Electrophoretic Mobility Shift Assay</subject><subject>Fundamental and applied biological sciences. Psychology</subject><subject>Fundamental immunology</subject><subject>Humans</subject><subject>Immunoglobulin Heavy Chains - metabolism</subject><subject>Immunoglobulin Light Chains - metabolism</subject><subject>Mice</subject><subject>Molecular immunology</subject><subject>Multiprotein Complexes - chemistry</subject><subject>Multiprotein Complexes - immunology</subject><subject>Multiprotein Complexes - metabolism</subject><subject>Proteomics</subject><subject>Receptors, Antigen, B-Cell - chemistry</subject><subject>Receptors, Antigen, B-Cell - immunology</subject><subject>Receptors, Antigen, B-Cell - metabolism</subject><subject>Receptors, Antigen, T-Cell - chemistry</subject><subject>Receptors, Antigen, T-Cell - metabolism</subject><subject>Stoichiometry</subject><subject>TCR</subject><subject>Techniques</subject><issn>0022-1759</issn><issn>1872-7905</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2007</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFkU1v1DAQhi0EotvCD-CCfAHBIcvY-XCsnlYVBaRCD8DZcpyJdlZJvNjeQv49Xu1KvcFpJOuZmXf8MPZKwFqAaD7s1jua1hJAraFeA5RP2Eq0ShZKQ_2UrQCkLISq9QW7jHEHAAIaeM4u8lvTNtCu2O8Nn22iB-R2TtT5fik6G7Hnk-9opLQUcUtD4gnddqZfB-Tvvm2-3n8vbIx2ec-T5z0mDBPNyNMWeUye3Jb8hCks3A98OoyJ9sEnpJk7P-1H_IPxBXs22DHiy3O9Yj9vP_64-Vzc3X_6crO5K1yl2lRI7aDWuhtsPgUG6F3VYKVEp7Qom7K1nRTYyFLbqoayAaiFdOXgalFpsNCVV-ztaW5OkNPHZCaKDsfRzugP0ShQILTU_wUlKCWrCjIoTqALPsaAg9kHmmxYjABz1GJ2JmsxRy0GapO15J7X5-GHbsL-sePsIQNvzoCNzo5DsLOj-Mi1GqSG4_LrE4f5zx4Ig4mOcHbYU0CXTO_pHzH-AqErqpY</recordid><startdate>20070731</startdate><enddate>20070731</enddate><creator>Swamy, Mahima</creator><creator>Minguet, Susana</creator><creator>Siegers, Gabrielle M.</creator><creator>Alarcón, Balbino</creator><creator>Schamel, Wolfgang W.A.</creator><general>Elsevier B.V</general><general>Elsevier</general><scope>IQODW</scope><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7T5</scope><scope>H94</scope><scope>7X8</scope></search><sort><creationdate>20070731</creationdate><title>A native antibody-based mobility-shift technique (NAMOS-assay) to determine the stoichiometry of multiprotein complexes</title><author>Swamy, Mahima ; Minguet, Susana ; Siegers, Gabrielle M. ; Alarcón, Balbino ; Schamel, Wolfgang W.A.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c478t-29c0599bfa0220f0dc46e471b7913638ab21e6239a4503600512c3fc51490a0b3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2007</creationdate><topic>Animals</topic><topic>Antibodies - metabolism</topic><topic>Biological and medical sciences</topic><topic>Blue Native</topic><topic>BN-PAGE</topic><topic>Cell Line</topic><topic>Cell Line, Tumor</topic><topic>Composition</topic><topic>Electrophoresis, Polyacrylamide Gel</topic><topic>Electrophoretic Mobility Shift Assay</topic><topic>Fundamental and applied biological sciences. Psychology</topic><topic>Fundamental immunology</topic><topic>Humans</topic><topic>Immunoglobulin Heavy Chains - metabolism</topic><topic>Immunoglobulin Light Chains - metabolism</topic><topic>Mice</topic><topic>Molecular immunology</topic><topic>Multiprotein Complexes - chemistry</topic><topic>Multiprotein Complexes - immunology</topic><topic>Multiprotein Complexes - metabolism</topic><topic>Proteomics</topic><topic>Receptors, Antigen, B-Cell - chemistry</topic><topic>Receptors, Antigen, B-Cell - immunology</topic><topic>Receptors, Antigen, B-Cell - metabolism</topic><topic>Receptors, Antigen, T-Cell - chemistry</topic><topic>Receptors, Antigen, T-Cell - metabolism</topic><topic>Stoichiometry</topic><topic>TCR</topic><topic>Techniques</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Swamy, Mahima</creatorcontrib><creatorcontrib>Minguet, Susana</creatorcontrib><creatorcontrib>Siegers, Gabrielle M.</creatorcontrib><creatorcontrib>Alarcón, Balbino</creatorcontrib><creatorcontrib>Schamel, Wolfgang W.A.</creatorcontrib><collection>Pascal-Francis</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Immunology Abstracts</collection><collection>AIDS and Cancer Research Abstracts</collection><collection>MEDLINE - Academic</collection><jtitle>Journal of immunological methods</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Swamy, Mahima</au><au>Minguet, Susana</au><au>Siegers, Gabrielle M.</au><au>Alarcón, Balbino</au><au>Schamel, Wolfgang W.A.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>A native antibody-based mobility-shift technique (NAMOS-assay) to determine the stoichiometry of multiprotein complexes</atitle><jtitle>Journal of immunological methods</jtitle><addtitle>J Immunol Methods</addtitle><date>2007-07-31</date><risdate>2007</risdate><volume>324</volume><issue>1</issue><spage>74</spage><epage>83</epage><pages>74-83</pages><issn>0022-1759</issn><eissn>1872-7905</eissn><coden>JIMMBG</coden><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 HC
2LC
2Igα/β
1 stoichiometry. Second, we demonstrate that the digitonin-extracted T cell antigen receptor (TCR) extracted from T cells has a stoichiometry of αβε
2γδζ
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.</abstract><cop>Amsterdam</cop><pub>Elsevier B.V</pub><pmid>17568608</pmid><doi>10.1016/j.jim.2007.05.003</doi><tpages>10</tpages></addata></record> |
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| subjects | Animals Antibodies - metabolism Biological and medical sciences Blue Native BN-PAGE Cell Line Cell Line, Tumor Composition Electrophoresis, Polyacrylamide Gel Electrophoretic Mobility Shift Assay Fundamental and applied biological sciences. Psychology Fundamental immunology Humans Immunoglobulin Heavy Chains - metabolism Immunoglobulin Light Chains - metabolism Mice Molecular immunology Multiprotein Complexes - chemistry Multiprotein Complexes - immunology Multiprotein Complexes - metabolism Proteomics Receptors, Antigen, B-Cell - chemistry Receptors, Antigen, B-Cell - immunology Receptors, Antigen, B-Cell - metabolism Receptors, Antigen, T-Cell - chemistry Receptors, Antigen, T-Cell - metabolism Stoichiometry TCR Techniques |
| title | A native antibody-based mobility-shift technique (NAMOS-assay) to determine the stoichiometry of multiprotein complexes |
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