Biology Reference
In-Depth Information
(400
M per labeling reaction) are diluted in dimethylformamide
(DMF) according to the manufacturer's instructions. Diluted
CyDyes are stored at −20°C and should be used within 3 months.
μ
4.
Lysine stock solution: 10 mM, stored at 4°C.
5.
Rehydration buffer: 8 M urea, 2% [w/v] CHAPS, a spatula tip
of bromophenol blue, DTT 20-100 mM (added directly
before usage), 0.5% [v/v] IPG buffer (added directly before
usage; corresponding to the IPG strip used for IEF (GE
Healthcare, Munich, Germany)). Rehydration buffer is stored
at −20°C.
3. Methods
3.1. Protein Extraction
with Phenol
This protocol is based on the protein extraction method according
to Hurkman and Tanaka (
19
) modifi ed by Colditz et al. (
20
).
Freshly ground samples are transferred to 2-mL Eppendorf vessels
and directly frozen in liquid nitrogen. For subsequent 2D DIGE
analysis, proteins from two different plant samples are extracted in
parallel.
1.
Take 200 mg pulverized plant material (see Note 2) from the
two protein fractions to be compared and add 750
μ
L extrac-
tion buffer. Incubate the samples for 10 min on ice.
2.
Add 750
L of water-saturated phenol (4°C) to each sample
and vortex. Incubate the samples for 30 min on a table mixer
(Eppendorf Thermomixer Compact; Eppendorf, Hamburg,
Germany) at 1,000 rpm at room temperature (see Note 3-5).
μ
3.
Centrifuge samples at 11,000 ×
g
for 10 min, 4°C.
4.
After centrifugation, transfer the phenol phase (generally the
upper phase) of each sample into a new 2-mL Eppendorf vessel
and dilute it in an equal volume of ice-cold extraction buffer.
Vortex samples (see Note 6).
5.
Centrifuge the samples at 11,000 ×
g
for 10 min, 4°C.
6.
Collect again the phenol phase (the upper phase) of each
sample and transfer it completely into a new preweighted 2-mL
Eppendorf vessel (see Note 7). Add ice-cold precipitation
solution up to a fi nal volume of 2 mL to each sample. Invert
the vessels several times back and forth and let the proteins
precipitate for at least 4 h at −20°C.
7.
Centrifuge the samples at 17,000 ×
g
for 3 min, 4°C. Discard
the supernatants and dilute the protein pellets in 1 mL of
ice-cold precipitation solution.
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