Detecting Kinase Activities from Single Cell Lysate Using Concentration-Enhanced Mobility Shift Assay
Electrokinetic preconcentration coupled with mobility shift assays can give rise to very high detection sensitivities. We describe a microfluidic device that utilizes this principle to detect cellular kinase activities by simultaneously concentrating and separating substrate peptides with different...
| Main Authors: | , , , , |
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| Other Authors: | |
| Format: | Article |
| Language: | en_US |
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American Chemical Society (ACS)
2015
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| Online Access: | http://hdl.handle.net/1721.1/100245 https://orcid.org/0000-0001-7215-1439 |
| _version_ | 1826201819506802688 |
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| author | Cheow, Lih Feng Sarkar, Aniruddh Kolitz, Sarah Han, Jongyoon Lauffenburger, Douglas A. |
| author2 | Massachusetts Institute of Technology. Department of Biological Engineering |
| author_facet | Massachusetts Institute of Technology. Department of Biological Engineering Cheow, Lih Feng Sarkar, Aniruddh Kolitz, Sarah Han, Jongyoon Lauffenburger, Douglas A. |
| author_sort | Cheow, Lih Feng |
| collection | MIT |
| description | Electrokinetic preconcentration coupled with mobility shift assays can give rise to very high detection sensitivities. We describe a microfluidic device that utilizes this principle to detect cellular kinase activities by simultaneously concentrating and separating substrate peptides with different phosphorylation states. This platform is capable of reliably measuring kinase activities of single adherent cells cultured in nanoliter volume microwells. We also describe a novel method utilizing spacer peptides that significantly increase separation resolution while maintaining high concentration factors in this device. Thus, multiplexed kinase measurements can be implemented with single cell sensitivity. Multiple kinase activity profiling from single cell lysate could potentially allow us to study heterogeneous activation of signaling pathways that can lead to multiple cell fates. |
| first_indexed | 2024-09-23T11:57:24Z |
| format | Article |
| id | mit-1721.1/100245 |
| institution | Massachusetts Institute of Technology |
| language | en_US |
| last_indexed | 2024-09-23T11:57:24Z |
| publishDate | 2015 |
| publisher | American Chemical Society (ACS) |
| record_format | dspace |
| spelling | mit-1721.1/1002452022-10-01T07:15:23Z Detecting Kinase Activities from Single Cell Lysate Using Concentration-Enhanced Mobility Shift Assay Cheow, Lih Feng Sarkar, Aniruddh Kolitz, Sarah Han, Jongyoon Lauffenburger, Douglas A. Massachusetts Institute of Technology. Department of Biological Engineering Massachusetts Institute of Technology. Department of Electrical Engineering and Computer Science Massachusetts Institute of Technology. Research Laboratory of Electronics Cheow, Lih Feng Sarkar, Aniruddh Kolitz, Sarah Lauffenburger, Douglas A. Han, Jongyoon Electrokinetic preconcentration coupled with mobility shift assays can give rise to very high detection sensitivities. We describe a microfluidic device that utilizes this principle to detect cellular kinase activities by simultaneously concentrating and separating substrate peptides with different phosphorylation states. This platform is capable of reliably measuring kinase activities of single adherent cells cultured in nanoliter volume microwells. We also describe a novel method utilizing spacer peptides that significantly increase separation resolution while maintaining high concentration factors in this device. Thus, multiplexed kinase measurements can be implemented with single cell sensitivity. Multiple kinase activity profiling from single cell lysate could potentially allow us to study heterogeneous activation of signaling pathways that can lead to multiple cell fates. National Institutes of Health (U.S.) (Grant P50-GM068762) Singapore. Agency for Science, Technology and Research (National Science Scholarship) 2015-12-14T20:14:13Z 2015-12-14T20:14:13Z 2014-07 2014-03 Article http://purl.org/eprint/type/JournalArticle 0003-2700 1520-6882 http://hdl.handle.net/1721.1/100245 Cheow, Lih Feng, Aniruddh Sarkar, Sarah Kolitz, Douglas Lauffenburger, and Jongyoon Han. “Detecting Kinase Activities from Single Cell Lysate Using Concentration-Enhanced Mobility Shift Assay.” Anal. Chem. 86, no. 15 (August 5, 2014): 7455–7462. https://orcid.org/0000-0001-7215-1439 en_US http://dx.doi.org/10.1021/ac502185v Analytical Chemistry Article is made available in accordance with the publisher's policy and may be subject to US copyright law. Please refer to the publisher's site for terms of use. application/pdf American Chemical Society (ACS) ACS |
| spellingShingle | Cheow, Lih Feng Sarkar, Aniruddh Kolitz, Sarah Han, Jongyoon Lauffenburger, Douglas A. Detecting Kinase Activities from Single Cell Lysate Using Concentration-Enhanced Mobility Shift Assay |
| title | Detecting Kinase Activities from Single Cell Lysate Using Concentration-Enhanced Mobility Shift Assay |
| title_full | Detecting Kinase Activities from Single Cell Lysate Using Concentration-Enhanced Mobility Shift Assay |
| title_fullStr | Detecting Kinase Activities from Single Cell Lysate Using Concentration-Enhanced Mobility Shift Assay |
| title_full_unstemmed | Detecting Kinase Activities from Single Cell Lysate Using Concentration-Enhanced Mobility Shift Assay |
| title_short | Detecting Kinase Activities from Single Cell Lysate Using Concentration-Enhanced Mobility Shift Assay |
| title_sort | detecting kinase activities from single cell lysate using concentration enhanced mobility shift assay |
| url | http://hdl.handle.net/1721.1/100245 https://orcid.org/0000-0001-7215-1439 |
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