Quantification of Protein Kinase Enzymatic Activity in Unfractionated Cell Lysates Using CSox‐Based Sensors
Defining perturbations in protein kinase activity within biological samples can provide insight into disease mechanisms as well as potential targets for drug development. In this article, we present a method that utilizes a phosphorylation-sensitive amino acid, termed CSox, to afford kinase-selectiv...
| Main Authors: | , , , |
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| Other Authors: | |
| Format: | Article |
| Language: | English |
| Published: |
Wiley
2020
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| Online Access: | https://hdl.handle.net/1721.1/126000 |
| _version_ | 1811086522024198144 |
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| author | Beck, Jon R. Peterson, Laura B Imperiali, Barbara Stains, Cliff I. |
| author2 | Massachusetts Institute of Technology. Department of Biology |
| author_facet | Massachusetts Institute of Technology. Department of Biology Beck, Jon R. Peterson, Laura B Imperiali, Barbara Stains, Cliff I. |
| author_sort | Beck, Jon R. |
| collection | MIT |
| description | Defining perturbations in protein kinase activity within biological samples can provide insight into disease mechanisms as well as potential targets for drug development. In this article, we present a method that utilizes a phosphorylation-sensitive amino acid, termed CSox, to afford kinase-selective biosensors capable of reporting on enzymatic activity directly in biological samples. These sensors produce an increase in fluorescence in response to phosphorylation of an amino acid residue adjacent to CSox. Probes can be designed for either serine/threonine or tyrosine kinases, and analysis can be performed using standard fluorescence equipment. The procedures provided herein represent our optimized protocols for the design, validation, and application of CSox-based protein kinase activity sensors. |
| first_indexed | 2024-09-23T13:27:16Z |
| format | Article |
| id | mit-1721.1/126000 |
| institution | Massachusetts Institute of Technology |
| language | English |
| last_indexed | 2024-09-23T13:27:16Z |
| publishDate | 2020 |
| publisher | Wiley |
| record_format | dspace |
| spelling | mit-1721.1/1260002022-09-28T14:22:39Z Quantification of Protein Kinase Enzymatic Activity in Unfractionated Cell Lysates Using CSox‐Based Sensors Beck, Jon R. Peterson, Laura B Imperiali, Barbara Stains, Cliff I. Massachusetts Institute of Technology. Department of Biology Massachusetts Institute of Technology. Department of Chemistry Defining perturbations in protein kinase activity within biological samples can provide insight into disease mechanisms as well as potential targets for drug development. In this article, we present a method that utilizes a phosphorylation-sensitive amino acid, termed CSox, to afford kinase-selective biosensors capable of reporting on enzymatic activity directly in biological samples. These sensors produce an increase in fluorescence in response to phosphorylation of an amino acid residue adjacent to CSox. Probes can be designed for either serine/threonine or tyrosine kinases, and analysis can be performed using standard fluorescence equipment. The procedures provided herein represent our optimized protocols for the design, validation, and application of CSox-based protein kinase activity sensors. National Institutes of Health (Grant NIH U54-CA112967) National Institutes of Health (Grant F32 GM102992) 2020-06-26T17:26:40Z 2020-06-26T17:26:40Z 2014-09 2019-12-09T21:40:57Z Article http://purl.org/eprint/type/JournalArticle 2160-4762 2160-4762 https://hdl.handle.net/1721.1/126000 Beck, John R. et al. "Quantification of Protein Kinase Enzymatic Activity in Unfractionated Cell Lysates Using CSox‐Based Sensors." Current Protocols 6, 3 (September 2014): 135-156 © 2014 John Wiley & Sons, Inc. en http://dx.doi.org/10.1002/9780470559277.ch140106 Current Protocols Creative Commons Attribution-Noncommercial-Share Alike http://creativecommons.org/licenses/by-nc-sa/4.0/ application/pdf Wiley PMC |
| spellingShingle | Beck, Jon R. Peterson, Laura B Imperiali, Barbara Stains, Cliff I. Quantification of Protein Kinase Enzymatic Activity in Unfractionated Cell Lysates Using CSox‐Based Sensors |
| title | Quantification of Protein Kinase Enzymatic Activity in Unfractionated Cell Lysates Using CSox‐Based Sensors |
| title_full | Quantification of Protein Kinase Enzymatic Activity in Unfractionated Cell Lysates Using CSox‐Based Sensors |
| title_fullStr | Quantification of Protein Kinase Enzymatic Activity in Unfractionated Cell Lysates Using CSox‐Based Sensors |
| title_full_unstemmed | Quantification of Protein Kinase Enzymatic Activity in Unfractionated Cell Lysates Using CSox‐Based Sensors |
| title_short | Quantification of Protein Kinase Enzymatic Activity in Unfractionated Cell Lysates Using CSox‐Based Sensors |
| title_sort | quantification of protein kinase enzymatic activity in unfractionated cell lysates using csox based sensors |
| url | https://hdl.handle.net/1721.1/126000 |
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