Determination of binding curves via protein micropatterning in vitro and in living cells
Quantification of protein interactions in living cells is of key relevance for understanding cellular signaling. With current techniques, however, it is difficult to determine binding affinities and stoichiometries of protein complexes in the plasma membrane. We introduce here protein micropatternin...
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| Veröffentlicht in: | Cytometry. Part A 2013-09, Vol.83 (9), p.847-854 |
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| container_issue | 9 |
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| container_title | Cytometry. Part A |
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| creator | Sunzenauer, Stefan Zojer, Verena Brameshuber, Mario Tröls, Andreas Weghuber, Julian Stockinger, Hannes Schütz, Gerhard J. |
| description | Quantification of protein interactions in living cells is of key relevance for understanding cellular signaling. With current techniques, however, it is difficult to determine binding affinities and stoichiometries of protein complexes in the plasma membrane. We introduce here protein micropatterning as a convenient and versatile method for such investigations. Cells are grown on surfaces containing micropatterns of capture antibody to a bait protein, so that the bait gets rearranged in the live cell plasma membrane. Upon interaction with the bait, the fluorescent prey follows the micropatterns, which can be readout with fluorescence microscopy. In this study, we addressed the interaction between Lck and CD4, two central proteins in early T‐cell signaling. Binding curves were recorded using the natural fluctuations in the Lck expression levels. Surprisingly, the binding was not saturable up to the highest Lck expression levels: on average, a single CD4 molecule recruited more than nine Lck molecules. We discuss the data in view of protein‐ and lipid‐mediated interactions. © 2012 International Society for Advancement of Cytometry |
| doi_str_mv | 10.1002/cyto.a.22225 |
| format | Article |
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We discuss the data in view of protein‐ and lipid‐mediated interactions. © 2012 International Society for Advancement of Cytometry</description><subject>Antibodies</subject><subject>Bacterial Proteins - genetics</subject><subject>CD4</subject><subject>CD4 Antigens - metabolism</subject><subject>Cell Line, Tumor</subject><subject>Cell Membrane - metabolism</subject><subject>equilibrium binding constant</subject><subject>HEK293 Cells</subject><subject>Humans</subject><subject>Lck</subject><subject>Luminescent Proteins - genetics</subject><subject>Lymphocyte Specific Protein Tyrosine Kinase p56(lck) - metabolism</subject><subject>Micropatterning</subject><subject>Microscopy, Fluorescence</subject><subject>plasma membrane</subject><subject>Protein Binding</subject><subject>Protein Interaction Mapping</subject><subject>single molecule microscopy</subject><issn>1552-4922</issn><issn>1552-4930</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2013</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqNkLtPwzAQxi0EoqWwMaOMDKT43WSsylNC6lIkmCzXviCjPEqcBPW_x2lKR8Qt99DvPt19CF0SPCUY01uzbaqpntIQ4giNiRA05inDx4ea0hE68_4TYyYwo6doRBmhgnA6Rm930EBduFI3riqjKovWrrSu_IhMW3fgo87paFNXDbgyKpypq41uwkbZI2HUuaauIl3avsldt9uEPPfn6CTTuYeLfZ6g14f71eIpflk-Pi_mL7FhMhXxemZJkoGUggtmEsukhgxmRHOGeQKSU2zsDIy1qdDcQAopS9ZaY0gsTWTGJuh60A1HfrXgG1U431-gS6har0j4XwqasvQfKGNUcsloQG8GNDzsfQ2Z2tSu0PVWEax621Vvu9JqZ3vAr_bK7boAe4B_fQ4AH4Bvl8P2TzG1eF8t54PuD2Qqj-E</recordid><startdate>201309</startdate><enddate>201309</enddate><creator>Sunzenauer, Stefan</creator><creator>Zojer, Verena</creator><creator>Brameshuber, Mario</creator><creator>Tröls, Andreas</creator><creator>Weghuber, Julian</creator><creator>Stockinger, Hannes</creator><creator>Schütz, Gerhard J.</creator><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>7X8</scope><scope>7QO</scope><scope>8FD</scope><scope>FR3</scope><scope>P64</scope></search><sort><creationdate>201309</creationdate><title>Determination of binding curves via protein micropatterning in vitro and in living cells</title><author>Sunzenauer, Stefan ; Zojer, Verena ; Brameshuber, Mario ; Tröls, Andreas ; Weghuber, Julian ; Stockinger, Hannes ; Schütz, Gerhard J.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c3695-b7d18fe665453c8d36aefe71a43048e6420cd7ecdd95a4ce9e938baa0e8d286f3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2013</creationdate><topic>Antibodies</topic><topic>Bacterial Proteins - genetics</topic><topic>CD4</topic><topic>CD4 Antigens - metabolism</topic><topic>Cell Line, Tumor</topic><topic>Cell Membrane - metabolism</topic><topic>equilibrium binding constant</topic><topic>HEK293 Cells</topic><topic>Humans</topic><topic>Lck</topic><topic>Luminescent Proteins - genetics</topic><topic>Lymphocyte Specific Protein Tyrosine Kinase p56(lck) - metabolism</topic><topic>Micropatterning</topic><topic>Microscopy, Fluorescence</topic><topic>plasma membrane</topic><topic>Protein Binding</topic><topic>Protein Interaction Mapping</topic><topic>single molecule microscopy</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Sunzenauer, Stefan</creatorcontrib><creatorcontrib>Zojer, Verena</creatorcontrib><creatorcontrib>Brameshuber, Mario</creatorcontrib><creatorcontrib>Tröls, Andreas</creatorcontrib><creatorcontrib>Weghuber, Julian</creatorcontrib><creatorcontrib>Stockinger, Hannes</creatorcontrib><creatorcontrib>Schütz, Gerhard J.</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>MEDLINE - Academic</collection><collection>Biotechnology Research Abstracts</collection><collection>Technology Research Database</collection><collection>Engineering Research Database</collection><collection>Biotechnology and BioEngineering Abstracts</collection><jtitle>Cytometry. 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With current techniques, however, it is difficult to determine binding affinities and stoichiometries of protein complexes in the plasma membrane. We introduce here protein micropatterning as a convenient and versatile method for such investigations. Cells are grown on surfaces containing micropatterns of capture antibody to a bait protein, so that the bait gets rearranged in the live cell plasma membrane. Upon interaction with the bait, the fluorescent prey follows the micropatterns, which can be readout with fluorescence microscopy. In this study, we addressed the interaction between Lck and CD4, two central proteins in early T‐cell signaling. Binding curves were recorded using the natural fluctuations in the Lck expression levels. Surprisingly, the binding was not saturable up to the highest Lck expression levels: on average, a single CD4 molecule recruited more than nine Lck molecules. We discuss the data in view of protein‐ and lipid‐mediated interactions. © 2012 International Society for Advancement of Cytometry</abstract><cop>United States</cop><pmid>23125142</pmid><doi>10.1002/cyto.a.22225</doi><tpages>8</tpages></addata></record> |
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| subjects | Antibodies Bacterial Proteins - genetics CD4 CD4 Antigens - metabolism Cell Line, Tumor Cell Membrane - metabolism equilibrium binding constant HEK293 Cells Humans Lck Luminescent Proteins - genetics Lymphocyte Specific Protein Tyrosine Kinase p56(lck) - metabolism Micropatterning Microscopy, Fluorescence plasma membrane Protein Binding Protein Interaction Mapping single molecule microscopy |
| title | Determination of binding curves via protein micropatterning in vitro and in living cells |
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