Abstract
Free-flow electrophoresis (FFE) is a technique for separation of proteins, peptides, organelles, and cells. With zone electrophoresis (ZE-FFE), organelles are separated according to surface charge. The ER is the only remaining major cellular compartment in Arabidopsis not to have been isolated using density centrifugation, immune-isolation, or any other method previously applied to purification of plant membranes. By using continuous-flow electrophoresis ER vesicles of similar surface charge, which may have been fragmented during cell lysis, can be focused. A large portion of these vesicles are of sufficiently different surface charge that separation from the majority of Golgi and other contaminants is possible. Here we adapt an earlier ZE-FFE Golgi isolation protocol for the isolation of highly pure ER vesicles and for tracking the migration of peripheral ER vesicles. Isolating ER vesicles of homogenous surface charge allows multi-'omic analyses to be performed on the ER. This facilitates investigations into structure–function relationships within the ER.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Dunkley TP et al (2006) Mapping the Arabidopsis organelle proteome. Proc Natl Acad Sci U S A 103:6518–6523
Agrawal GK et al (2011) Plant organelle proteomics: collaborating for optimal cell function. Mass Spectrom Rev 30:772–853
Drakakaki G et al (2012) Isolation and proteomic analysis of the SYP61 compartment reveal its role in exocytic trafficking in Arabidopsis. Cell Res 22:413–424
Kittelmann M, Hawes C, Hughes L (2016) Serial block face scanning electron microscopy and the reconstruction of plant cell membrane systems. J Microsc 263:200–211
Nixon-Abell J et al (2016) Increased spatiotemporal resolution reveals highly dynamic dense tubular matrices in the peripheral ER. Science 354(6311):aaf3928
Parsons HT, Fernández-Niño SM, Heazlewood JL (2014) Separation of the plant Golgi apparatus and endoplasmic reticulum by free-flow electrophoresis in plant proteomics. Methods Mol Biol 1072:527–539
Picotti P, Bodenmiller B, Aebersold R (2013) Proteomics meets the scientific method. Nat Methods 10:24–27
Aebersold R, Burlingame AL, Bradshaw RA (2013) Western blots versus selected reaction monitoring assays: time to turn the tables? Mol Cell Proteomics 12:2381–2382
Arike L, Peil L (2014) Spectral counting label-free proteomics. In: Martins-de-Souza D (ed) Shotgun proteomics: methods and protocols. Springer New York, New York, NY, pp 213–222
Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant 15:473–497
Dahl RH et al (2013) Engineering dynamic pathway regulation using stress-response promoters. Nat Biotechnol 31:1039–1046
Menges M, Murray JA (2002) Synchronous Arabidopsis suspension cultures for analysis of cell-cycle gene activity. Plant J 30:203–212
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2018 Springer Science+Business Media LLC
About this protocol
Cite this protocol
Parsons, H.T. (2018). Preparation of Highly Enriched ER Membranes Using Free-Flow Electrophoresis. In: Hawes, C., Kriechbaumer, V. (eds) The Plant Endoplasmic Reticulum . Methods in Molecular Biology, vol 1691. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7389-7_8
Download citation
DOI: https://doi.org/10.1007/978-1-4939-7389-7_8
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-7388-0
Online ISBN: 978-1-4939-7389-7
eBook Packages: Springer Protocols