Biology Reference
In-Depth Information
3.1.3 Combination
of TCA/Acetone
and Phenol-SDS
The fi rst step consists of preparing dry tissue powder (TCA/
acetone). Plant tissue is ground in liquid nitrogen in precooled
mortar and pestle to a fi ne powder: 200-400 mg of powder were
suspended in 1 mL of cold extraction buffer. Samples were vor-
texed thoroughly for 30 s, left at −20 °C for 30-60 min, and cen-
trifuged at 10,000 ×
g
for 5 min, at 4 °C. The supernatant was
discarded and the pellet was washed with cold acetone twice
(centrifuge at 10,000 ×
g
, for 3 min, at 4 °C). The pellet was dried
in vacuo. The second part of this protocol is protein extraction
with phenol-SDS. The pellet obtained is resuspended in 800
μ
L of
buffer phenol (Tris-buffered pH 8.0) added with 800
L SDS-Tris
buffer. Samples are mixed for at least 30 s and centrifuged for
3 min at 10,000 ×
g
. 300
μ
L upper-phase phenol is withdrawn, put
in new 2 mL microtube, 5 volumes of cold 0.1 M ammonium ace-
tate in methanol are added and kept for 30 min at −20 °C. After
centrifugation (10,000 ×
g
, 5 min), and the pellet is washed twice
with precipitation buffer II and twice with 80 % cold acetone. The
supernatant is decanted. Each time pellet should be re-suspended
completely and centrifuged at 10,000 ×
g
for 3 min at 4 °C. The
fi nal pellet is dried in vacuo.
μ
3.2 Protein
Labelling
Before starting the labelling, IPG strips have to be rehydrated.
450
L of rehydration solution containing ampholytes of pH 4-7
(24 cm) is placed in each lane of the reswelling tray. The IPG strip
can be placed in the lane with or without the light plastic cover
protecting the gel itself, with the gel side down. The gel is covered
with mineral oil to avoid drying and crystallization of urea. This
step has to be carried out overnight or at least for 10 h.
μ
1. The extracted proteins are resolubilized in the labelling solu-
tion. The high concentration of urea allows the denaturation
of proteins in order to obtain all the proteins present in one
conformation before their separation.
2. Reconstitution of CyDye with DMF: An anhydrous solution
of DMF (>99.8 % purity) has to be used (
see
Note 4
). To pre-
pare a stock solution of dyes, the adequate volume of DMF is
added to the tube containing the CyDye to reach the concen-
tration of 1 nmol/
μ
L. This solution may be kept for 2 months
at −20 °C.
3. The solution containing the proteins has to be equilibrated at
a pH between 8.0 and 9.0, the optimum pH for the reaction
between the CyDye and the proteins being 8.5. A fi rst test of
pH can be done with 0.5
L of solution on pH paper and the
adjustment is done by using NaOH solutions (0.1 or 1 M
depending on the starting pH of the protein solution).
4. Before the labelling and after the pH adjustment, the contents
of the protein sample have to be quantifi ed. Several methods
μ
