Biology Reference
In-Depth Information
1 .EXPERIMENTAL
DESIGN
2. SELECTION OF
PROTEIN EXTRACTION
CONDITIONS
3. SELECTION OF
CONDITIONS FOR 1-DE
IN LARGE GELS
(20X20)
4. SELECTION OF
CONDITIONS FOR 2-DE
IN LARGE GELS
(20X20)
DIFFERENTIAL
EVALUATION OF
RESPONSES TO
PATOGENS
PROTEIN YIELD
1-DE
2-DE
STAINING TREATMENT
PROTEIN QUANTITY
pH GRADIENT
Fig. 1
Workfl ow to select protein extraction and separation conditions from carnation roots and stems in pro-
teomics research
The proteomic comparative analysis, as any biological experiment,
must start with the experimental design. In the present work, car-
nation varieties with different levels of resistance to
F. oxysporum
f.
sp.
dianthi
, both inoculated and non-inoculated plants, with sam-
ples collected at different times after inoculation have been used
(
see
Note 1
). The plant material must be collected in a homoge-
neous way (
see
Note 2
). The infection and pathogen localization
must be confi rmed (
see
Note 3
).
3.1 Experimental
Design
3.2 Making the
Protein Extract
Three different protein extraction protocols reported in the litera-
ture were tested [
6
]. They include the use of treatments with TCA
and acetone to remove non-protein compounds and for protease
inactivation. The procedures applied to carnation stems and roots
are the following.
1. Transfer 20 mg of lyophilized plant material to a 2 mL
Eppendorf tube (
see
Note 4
).
2. Add 1 mL of total protein extraction buffer and shake for
30 min at 4 °C.
3. Centrifuge at 16,000 ×
g
for 10 min at 4 °C and transfer the
supernatant to a new 2 mL tube.
4. Add 1 mL of 100 % (w/v) TCA and mix well using vortex.
Stand for 2 h at 4 °C. Centrifuge at 16,000 ×
g
for 5 min at
4 °C. Discard the supernatant.
5. Wash the pellet (three times) with 80 % acetone.
6. Centrifuge at 16,000 ×
g
for 5 min at 4 °C and discard the
supernatant.
7. Dry the pellet to remove acetone residues in a fume
cupboard.
8. Dissolve the pellet in 200
3.2.1 Procedure Based
on Extraction with Buffer
and Subsequent TCA
Precipitation
μ
L of the solubilization solution and
shake for 4 h at 4 °C.
9. Quantify proteins using the Bradford method [
12
].
10. Store the extract at −20 °C for further analysis.
