MicroScale Thermophoresis (MST) for studying actin polymerization kinetics
Here, we present a MicroScale Thermophoresis (MST)–based assay for in vitro assessment of actin polymerization. By monitoring the thermophoretic behavior of ATTO488-labeled actin in a temperature gradient over time, we could follow polymerization in real time and resolve its three characteristic pha...
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| Language: | English |
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Taylor & Francis Group
2017-10-01
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| Series: | BioTechniques |
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| Online Access: | https://www.future-science.com/doi/10.2144/000114599 |
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| author | Andrea Topf Peter Franz Georgios Tsiavaliaris |
| author_facet | Andrea Topf Peter Franz Georgios Tsiavaliaris |
| author_sort | Andrea Topf |
| collection | DOAJ |
| description | Here, we present a MicroScale Thermophoresis (MST)–based assay for in vitro assessment of actin polymerization. By monitoring the thermophoretic behavior of ATTO488-labeled actin in a temperature gradient over time, we could follow polymerization in real time and resolve its three characteristic phases: nucleation, elongation, and steady-state equilibration. Titration experiments allowed us to evaluate the effects of actin-binding proteins (ABPs) on polymerization, including DNase I-induced inhibition and mDia2FH1FH2 (mDia2)-assisted acceleration of nucleation. The corresponding rates of actin filament elongation were quantitatively determined, yielding values in good agreement with those obtained using the pyrene-actin polymerization assay. Finally, we measured the effect of myosin on actin polymerization, circumventing the problems of fluorescence quenching and signal disturbance that occur with other techniques. MST is a simple and valuable research tool for investigating actin kinetics covering a wide range of molecular interactions, with low protein consumption. |
| format | Article |
| id | doaj-art-bcbbed87afe24e9cae46a7b348ffda59 |
| institution | OA Journals |
| issn | 0736-6205 1940-9818 |
| language | English |
| publishDate | 2017-10-01 |
| publisher | Taylor & Francis Group |
| record_format | Article |
| series | BioTechniques |
| spelling | doaj-art-bcbbed87afe24e9cae46a7b348ffda592025-08-20T02:25:58ZengTaylor & Francis GroupBioTechniques0736-62051940-98182017-10-0163418719010.2144/000114599MicroScale Thermophoresis (MST) for studying actin polymerization kineticsAndrea Topf0Peter Franz1Georgios Tsiavaliaris21Institute for Biophysical Chemistry, Hannover Medical School, Hannover, Germany1Institute for Biophysical Chemistry, Hannover Medical School, Hannover, Germany1Institute for Biophysical Chemistry, Hannover Medical School, Hannover, GermanyHere, we present a MicroScale Thermophoresis (MST)–based assay for in vitro assessment of actin polymerization. By monitoring the thermophoretic behavior of ATTO488-labeled actin in a temperature gradient over time, we could follow polymerization in real time and resolve its three characteristic phases: nucleation, elongation, and steady-state equilibration. Titration experiments allowed us to evaluate the effects of actin-binding proteins (ABPs) on polymerization, including DNase I-induced inhibition and mDia2FH1FH2 (mDia2)-assisted acceleration of nucleation. The corresponding rates of actin filament elongation were quantitatively determined, yielding values in good agreement with those obtained using the pyrene-actin polymerization assay. Finally, we measured the effect of myosin on actin polymerization, circumventing the problems of fluorescence quenching and signal disturbance that occur with other techniques. MST is a simple and valuable research tool for investigating actin kinetics covering a wide range of molecular interactions, with low protein consumption.https://www.future-science.com/doi/10.2144/000114599microscale thermophoresisactinpolymerizationactin-binding proteinkinetics |
| spellingShingle | Andrea Topf Peter Franz Georgios Tsiavaliaris MicroScale Thermophoresis (MST) for studying actin polymerization kinetics BioTechniques microscale thermophoresis actin polymerization actin-binding protein kinetics |
| title | MicroScale Thermophoresis (MST) for studying actin polymerization kinetics |
| title_full | MicroScale Thermophoresis (MST) for studying actin polymerization kinetics |
| title_fullStr | MicroScale Thermophoresis (MST) for studying actin polymerization kinetics |
| title_full_unstemmed | MicroScale Thermophoresis (MST) for studying actin polymerization kinetics |
| title_short | MicroScale Thermophoresis (MST) for studying actin polymerization kinetics |
| title_sort | microscale thermophoresis mst for studying actin polymerization kinetics |
| topic | microscale thermophoresis actin polymerization actin-binding protein kinetics |
| url | https://www.future-science.com/doi/10.2144/000114599 |
| work_keys_str_mv | AT andreatopf microscalethermophoresismstforstudyingactinpolymerizationkinetics AT peterfranz microscalethermophoresismstforstudyingactinpolymerizationkinetics AT georgiostsiavaliaris microscalethermophoresismstforstudyingactinpolymerizationkinetics |