The group of Simone Reber at IRI Life Sciences has been invited to publish two articles in STAR Protocols, which are connected to their publication in Current Biology earlier this year.*
Cytoplasmic extracts from unfertilised Xenopus eggs have made important contributions to our understanding of microtubule dynamics, spindle assembly, and scaling. Until recently, these in vitro studies relied on the use of heterologous tubulin.
This protocol details the purification of tubulin from egg extracts of three different Xenopus species, which yields milligrams of physiologically relevant tubulins. The purified tubulin is a complex mixture of isoforms with various post-translational modifications specific to the source, i. e. eggs of different frog species.
The protocol is applicable to any cell or tissue of interest.
Imaging microtubule dynamics can be instructive for a given species, isoform composition or biochemical modification.
Here, we describe step by step two methods that visualize microtubule dynamics at high speed and high contrast:
(1) Total internal reflection fluorescence microscopy (TIRFM)
(2) label-free interference reflection microscopy (IRM).
* Original publication: Hirst WG, Biswas A, Mahalingan KK and Reber S. (2020) “Differences in intrinsic tubulin dynamic properties contribute to spindle length control in Xenopus species”.