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Western blotting of the sample and probing available antibodies
of known subcellular localized proteins such as RubisCO as a chloro-
plast marker, KAT2 as a peroxisome marker, and porin as a mitochon-
drial marker can also assess the purity of isolated mitochondria.
2D-IEF-SDS-DIGE can be used to compare the purity of mito-
chondrial fractions by identifying the proteins that differ in
abundance between two samples that were prepared using dif-
ferent isolation procedures. We have previously used 2D-IEF-
SDS-DIGE to show that FFE purifi cation of mitochondria
results in lower abundance of proteins from contaminating
organelles (Fig.
2
, [
9
,
28
]). Compared to other methods of
assessing mitochondrial purity such as Western blot, the main
advantage of 2D-IEF- SDS-DIGE is that a far higher number of
proteins can be quantifi ed in a single experiment (Fig.
2
).
3.4.4 2D-IEF-SDS
Differential In-Gel
Electrophoresis of Purifi ed
Mitochondrial Proteins
Fig.
2
Example of 2D-IEF-SDS-DIGE of isolated rice mitochondria before and
after free fl ow electrophoresis purifi cation to determine the degree of contami-
nation [
28
]. Samples before FFE treatment (−FFE; labelled with Cy3, shown in
red
) and after FFE treatment (+FFE; labelled with Cy5, shown in
green
) were
compared. The
top panels
are gel images of each fl uorescence signal, and the
bottom panel
is a combined fl uorescence image electronically overlaid using
ImageQuant TL software (GE Healthcare Life Sciences).
Yellow spots
represent
proteins of equal abundance before and after FFE purifi cation. Spots that are
more abundant in samples before FFE purifi cation are
red
, and those more abun-
dant in samples after FFE purifi cation are
green
. The
numbered arrows
indicate
proteins with statistically signifi cantly decreased abundance after FFE purifi ca-
tion (
n
= 3,
P
> 0.05), which were chloroplast or peroxisome proteins when iden-
tifi ed by mass spectrometry
