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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| Main Authors: | , , |
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| Format: | Article |
| Language: | English |
| Published: |
Taylor & Francis Group
2017-10-01
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| Series: | BioTechniques |
| Subjects: | |
| Online Access: | https://www.future-science.com/doi/10.2144/000114599 |
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| Summary: | 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. |
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| ISSN: | 0736-6205 1940-9818 |