High-Throughput Protein-Nucleic Acid Interaction Assay Based on Protein-Induced Fluorescence Enhancement
Molecular processes involved in gene expression encompass multitudes of interactions between proteins and nucleic acids. Quantitative description of these interactions is crucial for delineating the mechanisms governing transcription, genome duplication, and translation. Here we describe a detailed protocol for the quantitative analysis of protein-nucleic acid interactions based on protein-induced fluorescence enhancement (PIFE). While PIFE has mainly been used in single-molecule studies, we modified its application for bulk measurement of protein-nucleic acid interactions in microwell plates using standard fluorescent plate readers. The microwell plate PIFE assay (mwPIFE) is simple, does not require laborious protein labeling, and is high throughput. These properties predispose mwPIFE to become a method of choice for routine applications that require multiple parallel measurements such as buffer optimization, competition experiments, or screening chemical libraries for binding modulators.
| Medienart: |
E-Artikel |
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| Erscheinungsjahr: |
2021 |
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| Erschienen: |
2021 |
| Enthalten in: |
Zur Gesamtaufnahme - volume:2209 |
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| Enthalten in: |
Methods in molecular biology (Clifton, N.J.) - 2209(2021) vom: 17., Seite 109-117 |
| Sprache: |
Englisch |
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| Beteiligte Personen: |
Fulneček, Jaroslav [VerfasserIn] |
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| Links: |
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| Themen: |
63231-63-0 |
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| Anmerkungen: |
Date Completed 31.03.2021 Date Revised 31.03.2021 published: Print Citation Status MEDLINE |
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| doi: |
10.1007/978-1-0716-0935-4_7 |
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| funding: |
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| Förderinstitution / Projekttitel: |
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| PPN (Katalog-ID): |
NLM317687492 |
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| 520 | |a Molecular processes involved in gene expression encompass multitudes of interactions between proteins and nucleic acids. Quantitative description of these interactions is crucial for delineating the mechanisms governing transcription, genome duplication, and translation. Here we describe a detailed protocol for the quantitative analysis of protein-nucleic acid interactions based on protein-induced fluorescence enhancement (PIFE). While PIFE has mainly been used in single-molecule studies, we modified its application for bulk measurement of protein-nucleic acid interactions in microwell plates using standard fluorescent plate readers. The microwell plate PIFE assay (mwPIFE) is simple, does not require laborious protein labeling, and is high throughput. These properties predispose mwPIFE to become a method of choice for routine applications that require multiple parallel measurements such as buffer optimization, competition experiments, or screening chemical libraries for binding modulators | ||
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