JoVE Journal

Biochemistry

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Purification and Quality Control of Recombinant Septin Complexes for Cell-Free Reconstitution

2.0K Views

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11:50 min

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June 23rd, 2022

DOI :

10.3791/63871-v

June 23rd, 2022

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Gerard Castro-Linares¹, Jeffrey den Haan¹, Francois Iv², Carla Silva Martins², Aurélie Bertin³, Manos Mavrakis², Gijsje H. Koenderink¹

¹Department of Bionanoscience, Kavli Institute of Nanoscience Delft, Delft University of Technology, ²Institut Fresnel, CNRS UMR7249, Aix Marseille Univ, Centrale Marseille, ³Institut Curie, Université PSL, Sorbonne Université

Summary

In vitro reconstitution of cytoskeletal proteins is a vital tool to understand the basic functional properties of these proteins. The present paper describes a protocol to purify and assess the quality of recombinant septin complexes, which play a central role in cell division and migration.

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Quality Control

Recombinant Septin Complexes

Cell free Reconstitution

Addgene Plasmids

Protein Expression

Affinity Chromatography

His tagged Proteins

Strep II Tagged Proteins

Nickel Sepharose

Protein Liquid Chromatography

Chapters in this video

0:05

Introduction

0:47

Purification of Septin Hetero-Oligomers

4:29

Quality Control of Purity and Integrity of the Septin Hetero-Oligomer

7:34

Septin Functional Quality Control via Polymerization Analysis

8:57

Results: Analysis of Purified Septin Quality

11:01

Conclusion

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