Measurement of tubulin oligomers self-assembly by FRET.
Microtubules nucleation, dynamic instability and mechanical properties are regulated by tubulin-tubulin longitudinal and lateral interactions. It is thus crucial to develop assays that can investigate potential modifications of these interactions due to tubulin post-translational modifications or mutations. Here we describe a detailed protocol for a method to monitor tubulin oligomerization at a step before MT nucleation occurs, using a FRET-based assay. Briefly, the principle of the technique is to measure over time the fluorescence emitted by a FRET pair of fluorophores when tubulin dimers interact with each other below the critical concentration for microtubules self-assembly. This technique can also be used to monitor more specifically lateral interactions between protofilaments each labelled with one FRET pair of fluorophores. The experiment can be completed within 2-3 hours.
Posted 09 Mar, 2017
Measurement of tubulin oligomers self-assembly by FRET.
Posted 09 Mar, 2017
Microtubules nucleation, dynamic instability and mechanical properties are regulated by tubulin-tubulin longitudinal and lateral interactions. It is thus crucial to develop assays that can investigate potential modifications of these interactions due to tubulin post-translational modifications or mutations. Here we describe a detailed protocol for a method to monitor tubulin oligomerization at a step before MT nucleation occurs, using a FRET-based assay. Briefly, the principle of the technique is to measure over time the fluorescence emitted by a FRET pair of fluorophores when tubulin dimers interact with each other below the critical concentration for microtubules self-assembly. This technique can also be used to monitor more specifically lateral interactions between protofilaments each labelled with one FRET pair of fluorophores. The experiment can be completed within 2-3 hours.
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