Biology Reference
In-Depth Information
Chapter 7
A Protocol for Protein Extraction from Lipid-Rich Plant
Tissues Suitable for Electrophoresis
Agnieszka Zienkiewicz , Juan David Rejón , Juan de Dios Alché ,
María Isabel Rodríguez-García , and Antonio Jesús Castro
Abstract
Plant tissues contain high levels of nonprotein contaminants such as lipids, phenolic compounds, and
polysaccharides among others, which interfere with protein extraction and electrophoretic separation.
Preparation of good-quality protein extracts is a critical issue for successful electrophoretic analysis. Here,
we describe a three-step method for protein extraction from lipid-rich plant tissues, which is suitable for
both 1-D and 2-D electrophoresis and is compatible with downstream applications. The protocol includes
prefractionation, fi ltration, and TCA/acetone precipitation steps prior to protein resolubilization.
Key words
Lipid removal, Lipid-rich plant tissues, Protein extraction, TCA/acetone precipitation,
Two-dimensional electrophoresis
1
Introduction
Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE)
is a powerful and widely used method to study complex mixtures
of hundreds or even thousands of different proteins extracted from
cells, tissues, or complete organisms [
1
-
3
]. Proteins are separated
on a 2-D matrix on the basis of their charge (IEF, fi rst dimension)
and size (SDS-PAGE, second dimension) [
1
]. Sample preparation
is a critical factor for achieving good and reproducible 2D-PAGE
results. Protein extraction from plant tissues is challenging due to
structural (i.e., presence of a cell wall) and chemical (i.e., produc-
tion of a broad spectrum of metabolites such as pigments, lipids,
phenolic compounds, polysaccharides) reasons [
4
-
9
].
Oleaginous plants (e.g., sunfl ower, olive, rapeseed) accumulate
high amounts of storage neutral lipids (e.g., TAGs) for energy sup-
ply during periods of active metabolism [
10
]. These compounds
are confi ned to specialized organelles called oil bodies [
11
], which
can be found in different plant tissues like seeds [
12
], anthers [
13
],
