Biology Reference
In-Depth Information
Mw Markers
(KDa)
Mw Markers
(KDa)
1 23456
123456
200
116
97
200
116
97
66
66
45
45
31
31
22
22
14
14
6.5
6.5
Number of
bands
54
56
58
56
56 56
Fig. 4
Staining comparison protocols. SYPRO Ruby (
left
) and SYPRO
Ruby + Coomasie colloidal (
right
). Analysis in 1-DE (20 × 20 cm) for protein
extracts from carnation roots infected with
Fod
in different hours post-inocula-
tion (hpi). Resistant control 0 hpi (1), Susceptible control 0 hpi (2), Resistant
control 6 hpi (3), Resistant inoculated 6 hpi (4), Susceptible control 6 hpi (5),
Susceptible inoculated 6 hpi (6). The number of bands for treatment 1, 3, and 4
is shown in the
bottom
of the graph
3.6 Two-Dimensional
Gel Electrophoresis on
Large Strips
Perform 2-DE of different amounts of protein in the range of
200-400
g for 17 cm-long IPG strips (pH 3-10). In order to
evaluate the separation resolution for the fi rst dimension, compare
lineal and no lineal IPG strips (pH 3-10) (
see
Note 18
).
μ
1. Add protein extract obtained from the tissue under study in
equivalent amounts of 200 and 400
3.6.1
Amount of Protein
g of protein in different
1.5 mL tubes. Add IPG strip rehydration solution to a fi nal vol-
ume of 400
μ
2. Pour gently the previously prepared mixture creating a line in
one of the rails of the IPG strip focusing tray, avoiding the
formation of bubbles. Repeat the procedure for each sample.
3. Place in each rail a 17 cm-long IPG strip with a 3-10 pH linear
range, ensuring that it is properly placed to avoid the entry of
air bubbles between the strip and the sample. The position of
the strip must allow its end marks to coincide with the position
of the (+, anode) and (−, cathode) electrodes.
4. Add 1-2 mL of mineral oil to prevent dehydration during the
rehydration process and cover with the plastic faceplate of the
system.
5. Perform the rehydration process placing the IPG strip focus-
ing tray in the focusing system for 16 h applying 50 V at 20 °C
(active rehydration).
μ
L. Shake vigorously by vortexing (
see
Note 11
).
