Chapter title |
Preparation of Highly Enriched ER Membranes Using Free-Flow Electrophoresis
|
---|---|
Chapter number | 8 |
Book title |
The Plant Endoplasmic Reticulum
|
Published in |
Methods in molecular biology, January 2018
|
DOI | 10.1007/978-1-4939-7389-7_8 |
Pubmed ID | |
Book ISBNs |
978-1-4939-7388-0, 978-1-4939-7389-7
|
Authors |
Harriet T. Parsons, Parsons, Harriet T. |
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. |
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