Biology Reference
In-Depth Information
Antibody Marker
cFBPase
Cytosol
pMDH2
Peroxisome
RbcL
Plastid
Mitochondria
VDAC-1
Histone H3
Nucleus
Calreticulin
Endomembrane
Fig. 2
Immunological analysis of the cytosolic enrichment method using 5 µg
protein from each fraction. Western blotting of subcellular protein markers from
Arabidopsis
cell culture protoplasts (protoplast), 10,000 ×
g
crude mixed organ-
elle pellet (10K pellet), 100,000 ×
g
crude mixed organelle pellet (100K pellet)
and cytosolic fraction (cytosol) after SDS-PAGE. Polyclonal antibodies rose
against
Arabidopsis
cFBPase (cytosol), pMDH2 (peroxisome), RbcL (plastid),
VDAC-1 (mitochondria), histone H3 (nucleus), and calreticulin (endomembrane
system) are applied to the samples to determine their enrichment of subcellular
compartments. The lack of organelle protein contaminants in the cytosolic frac-
tion is clearly seen
Diafi ltrate the cytosolic fraction with 100 % H
2
O and
concentrate sample using 5 kDa Ultrafree centrifugal fi lter
device (
see
Note 10
).
(d) Perform total protein quantifi cation with Bradford or
Lowry assay. Immediately store protein samples in 2 mg
aliquots at −80 °C.
4. Purity assessment
(a) Purity of the cytosolic fraction can be assessed by immu-
noblotting with commercially available antibodies against
plant organelle protein markers (e.g., mitochondria, plas-
tids, nuclei, the ER, plasma membranes, peroxisomes, and
cytosol) (Fig.
2
).
(b) In addition, mass spectrometry-based selected reaction
monitoring (SRM) can be used to quantify organelle
protein contamination in the cytosolic fraction as a sup-
plement to immunoblotting. SRM is a highly specifi c and
sensitive tandem mass spectrometry technique for quanti-
fying an individual precursor ion and its fragment ion
(transition) in a complex biological mixture. A selection of
SRM transitions have been established for profi ling the
cytosolic fraction after enrichment [
10
].
