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Related Experiment Video

Updated: Jan 26, 2026

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Purification of tubulin with controlled post-translational modifications by polymerization-depolymerization cycles.

Judith Souphron1,2, Satish Bodakuntla1,2, A S Jijumon1,2

  • 1Institut Curie, PSL Research University, CNRS UMR 3348, Orsay, France.

Nature Protocols
|April 19, 2019
PubMed
Summary

Researchers developed a new protocol to purify tubulin with controlled post-translational modifications (PTMs) for in vitro microtubule studies. This method enables analysis of the "tubulin code" and microtubule dynamics using functional tubulin variants.

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Area of Science:

  • Cell Biology
  • Biochemistry
  • Structural Biology

Background:

  • Microtubule dynamics are crucial for cellular functions and are regulated by the "tubulin code," involving tubulin isotypes and post-translational modifications (PTMs).
  • Previous in vitro reconstitution experiments were limited by the use of heavily modified tubulin purified from brain tissue.
  • Analyzing diverse tubulin variants in vitro is essential for understanding the tubulin code mechanism.

Purpose of the Study:

  • To develop a novel protocol for purifying tubulin with controlled PTMs from various cell sources.
  • To enable in vitro reconstitution experiments investigating microtubule dynamics and protein interactions.
  • To provide a method for obtaining functional tubulin variants for mechanistic studies.

Main Methods:

  • A protocol involving cycles of polymerization and depolymerization was developed for tubulin purification.
  • The method allows purification from adherent cells, suspension cultures, and limited tissue sources (e.g., single mouse brains).
  • Purification can be combined with cell treatments (drug, transfection) or enzymatic modifications.

Main Results:

  • The protocol yields highly pure tubulin, excluding non-polymerizable variants, thus selecting for functional tubulin.
  • It allows for controlled PTMs, essential for studying the tubulin code.
  • Purified tubulin is suitable for advanced assays like total internal reflection fluorescence (TIRF) microscopy and pelleting assays.

Conclusions:

  • A novel, efficient protocol for purifying functional tubulin with controlled PTMs has been established.
  • This method facilitates in vitro studies of microtubule dynamics and the tubulin code.
  • The protocol supports diverse applications in cell biology and biochemistry, advancing our understanding of microtubule regulation.