Biology Reference
In-Depth Information
plant tissues is to minimize the cross-contamination of chloroplasts
with the intact protoplasts. Density gradients have been demon-
strated to prevent such contamination [
26
].
Here, we present two techniques for the purifi cation of cyto-
solic enriched fractions from
Arabidopsis
. The methods are based
on published procedures, which have been adapted to isolate cyto-
solic enriched fractions from either
Arabidopsis
cell cultures [
19
,
20
]
or seedlings [
23
,
28
]. These procedures have been successfully
used for large scale proteomic characterization of the cytosolic
fraction from cultured cells [
10
] or for the study of protein local-
ization between cytosol and chloroplasts of
Arabidopsis
seedlings
[
26
]. The isolated fractions can be cleared of other cellular con-
taminants via differential centrifugation and sugar gradients. In
addition to the cytosolic fraction of seedlings, chloroplasts can also
be isolated using a discontinuous Percoll™ gradient after proto-
plastation [
12
]. The fi nal fractions can be analyzed for organelle
contamination by immunoblotting or mass spectrometry-based
selected reaction monitoring against known subcellular protein
markers. We also discuss how to optimize the procedure to varying
conditions that affect the purity of the fractions (e.g., the age and
the quality of the starting material or the type of homogenizer and
frequency of strokes).
2
Materials
Prepare all solutions using ultrapure water (prepared by purifying
deionized water to attain a sensitivity of 18 M
cm at 25 °C) and
analytical grade reagents. Prepare reagents at room temperature.
Perform all centrifugation steps at 4 °C.
Ω
2.1 Purifi cation
of the Cytosolic
Fraction from Plant
Cell Cultures
1. Plant material:
Arabidopsis
cell culture.
2. Enzyme Buffer: 0.4 M mannitol, 3.5 mM MES-NaOH buffer,
pH 5.7, 0.4 % (w/v) cellulase RS (Yakult Pharmaceutical,
Tokyo, Japan) and 0.05 % (w/v) pectolyase Y-23 (Yakult
Pharmaceutical, Tokyo, Japan). Prepare immediately before
use (
see
Note 1
).
3. Wash Buffer: 0.4 M mannitol, 3.5 mM MES-NaOH, pH 5.7.
4. Homogenization Buffer: 0.4 M sucrose (osmotic), 3 mM
EDTA, 50 mM Tris-HCl buffer and 2 mM dithiothreitol
[DTT]. Add reducing agent DTT just prior to homogeniza-
tion (
see
Note 2
).
5. Glass-Tefl on Potter-Elvehjem Tissue Grinders (30-50 mL
capacity), cooled on ice. For gentle disruption of protoplasts,
the space between the Tefl on pestle and the tube should be
approximately 100
m.
6. Miracloth (Merck KGaA, Germany).
μ
