In vitro preparation of single filamentous microtubules optimized for dynamic and electrophoretic light scattering measurements. v1

The lack of essential information on sample preparation and the need to characterize and understand microtubules (MTs) in various biological functionalities within the cell have led us to develop a high-quality polymerization protocol to prepare fully formed MTs in vitro, which is particularly designed for Dynamic light scattering (DLS) and Electrophoretic light scattering (ELS) experiments for further analysis. This protocol details the reconstitution of tubulin, GTP, and Taxol, the preparation of PEM, cushion buffer, and polymerization buffers, the step-by-step polymerization process, and the use of Taxol to stabilize the microtubule filaments without precipitation. This polymerization protocol successfully generated samples for light scattering experiments using buffers that replicate physiological conditions. This has provided consistency in preparing stable, diluted, aggregate-free, homogenous microtubule filament samples that could benefit many other scientific research groups currently working in the field. Additionally, it can easily be adapted to prepare samples using other buffers and biological fluids. Graphical abstract: This sample preparation protocol requires 3 days. All the buffers and solutions are prepared to make enough of four samples of fully developed microtubules in 100 millimolar concentration of potassium chloride (KCl), with a total volume of 1 milliliter of solution. (See Materials sections).