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Enrichment of Golgi Membranes from Triticum aestivum (Wheat) Seedlings

  • Wei Zeng
  • Berit Ebert
  • Harriet T. Parsons
  • Carsten Rautengarten
  • Antony Bacic
  • Joshua L. HeazlewoodEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1511)

Abstract

The Golgi apparatus is an essential component in the plant secretory pathway. The enrichment of Golgi membranes from plant tissue is fundamental to the study of this structurally complex organelle. The utilization of density centrifugation for the enrichment of Golgi membranes is still the most widely employed isolation technique. Generally, the procedure requires optimization depending on the plant tissue being employed. Here we provide a detailed enrichment procedure that has previously been used to characterize cell wall biosynthetic complexes from wheat seedlings. We also outline several downstream analyses procedures, including nucleoside diphosphatase assays, immunoblotting, and finally localization of putative Golgi proteins by fluorescent tags.

Key words

Golgi apparatus Density gradient centrifugation Subcellular localization Nucleoside diphosphatase 

Notes

Acknowledgments

This work was funded by grants from the Australia Research Council (ARC) to the ARC Centre of Excellence in Plant Cell Walls [CE110001007] and the U. S. Department of Energy, Office of Science, Office of Biological and Environmental Research, through contract DE-AC02-05CH11231 between Lawrence Berkeley National Laboratory and the U. S. Department of Energy. JLH is supported by an ARC Future Fellowship [FT130101165].

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Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  • Wei Zeng
    • 1
  • Berit Ebert
    • 1
    • 2
  • Harriet T. Parsons
    • 3
  • Carsten Rautengarten
    • 1
    • 2
  • Antony Bacic
    • 1
  • Joshua L. Heazlewood
    • 3
    • 4
  1. 1.The ARC Centre of Excellence in Plant Cell Walls, School of BiosciencesThe University of MelbourneMelbourneAustralia
  2. 2.Department of BiochemistryUniversity of CambridgeCambridge CB2 1QRUK
  3. 3.Joint BioEnergy Institute and Biological Systems and Engineering DivisionLawrence Berkeley National LaboratoryBerkeleyUSA
  4. 4.School of BioSciencesThe University of MelbourneMelbourneAustralia

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