Biology Reference
In-Depth Information
Chapter 35
Separation of the Plant Golgi Apparatus and Endoplasmic
Reticulum by Free-Flow Electrophoresis
Harriet T. Parsons , Susana M. González Fernández-Niño ,
and Joshua L. Heazlewood
Abstract
Free-fl ow 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 plant
Golgi and endoplasmic reticulum (ER) are similar in density and are therefore separated with diffi culty
using standard techniques such as density centrifugation. Purifi cation of the ER and Golgi apparatus per-
mits a biochemical and proteomic characterization which can reveal the division of processes between these
compartments. Here we describe complete separation between the ER and more negatively charged Golgi
compartments using ZE-FFE. We also describe techniques for assigning proteins to partially separated ER
and the less negatively charged Golgi compartments.
Key words
Free-fl ow electrophoresis, Golgi apparatus, Arabidopsis, Endoplasmic reticulum,
Proteomics
1
Introduction
Zone electrophoresis employing a free-fl ow electrophoresis system
(ZE-FFE) has existed for over 50 years. Its potential application to
subcellular fractionation can be fully realized when the process is
combined with modern mass spectrometry analytical techniques.
Its resurgence as a technique across multiple research domains is
therefore relatively recent considering its availability for the past
half century.
ZE-FFE is typically applied to samples enriched in the subcellu-
lar compartment of interest, usually via density centrifugation. This
technique has advanced subcellular proteomics in plants through
isolation of organelles at appreciably higher purity levels than
achieved previously with density centrifugation techniques alone
[
1
,
2
]. The separation of mitochondria from peroxisomes [
3
,
4
] and
purifi cation of Golgi compartments from a complex background of
