Biology Reference
In-Depth Information
Fig. 1
Number of plant proteomics publications till March 2012 reported in ISI Topic = (proteomics and “species”)
2
Protein Extraction
Most plant tissues are not a ready source for protein extraction and
need specifi c precautions. The cell wall and the vacuole make up
the majority of the cell mass. Consequently, plant tissues have a
relatively low protein content. Moreover, the cell wall and the vac-
uole are associated with numerous substances responsible for irre-
producible results such as proteolytic breakdown and charge
heterogeneity. Compared to Arabidopsis, several orphan species
contain extremely high levels of interfering compounds. The
majority of the plant protocols introduce a precipitation step to
concentrate the proteins and to separate them from the interfering
compounds. The protein precipitation step can be preceded by a
denaturing or non-denaturing extraction step and is combined
with one or two washing steps to remove introduced salt ions and
other remaining interfering substances. The most commonly used
method for extraction of plant proteins is the trichloroacetic acid
(TCA)/acetone precipitation method [
5
]. Proteins are very sensi-
tive to denaturation at low pH. TCA is a strong acid (p
K
a
0.7) that
is soluble in organic solvents. We have tested in the past different
protocols to extract proteins from plant samples [
6
]. In our hands
and also according to others [
7
,
8
], the phenol extraction protocol
proved to be the most powerful for recalcitrant plant tissues. The
protein precipitation step is in this method achieved by ammonium
acetate and methanol and is preceded by a denaturing phenol
extraction. Moreover, the low pH of TCA might create problems
with basic chemical labeling methods. For a more detailed discus-
sion of protein extraction the reader is referred to [
6
,
9
,
10
].
