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Purification of Tubulin with Controlled Posttranslational Modifications and Isotypes from Limited Sources by Polymerization-Depolymerization Cycles
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Bioquímica
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JoVE Journal Bioquímica
Purification of Tubulin with Controlled Posttranslational Modifications and Isotypes from Limited Sources by Polymerization-Depolymerization Cycles

Purification of Tubulin with Controlled Posttranslational Modifications and Isotypes from Limited Sources by Polymerization-Depolymerization Cycles

DOI:
10.3791/61826-v

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07:54 min

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November 05, 2020

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, , ,
1PSL Research University, CNRS UMR3348,Institut Curie, 2Université Paris-Saclay, CNRS UMR3348,Université Paris Sud

Capítulos

  • 00:05Introduction
  • 00:34Amplification and Lysis of Cells in Suspension Cultures
  • 02:08Amplification and Lysis of Adherent Cells and Mouse Brain Tissue Lysis
  • 03:46Sample Preparation for Analysis
  • 04:10Clarification, Polymerization, and Depolymerization of the Lysate
  • 06:27Evaluation, Quantification, and Characterization of Purified Tubulin
  • 07:19Conclusion

Summary

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This protocol describes tubulin purification from small/medium-scale sources such as cultured cells or single mouse brains, using polymerization and depolymerization cycles. The purified tubulin is enriched in specific isotypes or has specific posttranslational modifications and can be used in in vitro reconstitution assays to study microtubule dynamics and interactions.

Tags

Keywords: Tubulin PurificationPosttranslational ModificationsIsotypesPolymerization-depolymerization CyclesSuspension CulturesAdherent Cell CulturesCell HarvestingLysis BufferPBS-EDTA

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