Biology Reference
In-Depth Information
3-10 LINEAL
3-10 NON LINEAL
pH 3
pH10
pH 3
pH10
200
116
97
200
116
97
66
45
66
45
31
31
22
22
14
14
6.5
6.5
Fig. 5
Two-DE in large gels (20 × 20 cm) of extracts obtained from carnation stems using different pH gradient
in the fi rst dimension separation:
left
(Lineal) and
right
(non lineal)
19. Capture the image using the calibrated densitometer (GS-800
Calibrated Densitometer, Bio-Rad).
20. Compare visually the resolution and the number of present
spots for both amounts of evaluated proteins, in each organ of
the plant (
see
Note 19
).
1. Add in different 1.5 mL tubes extract equivalent to 400
μ
g of
3.6.2 pH Gradient
L using IPG strip rehydration
solution. Shake vigorously by vortexing (
see
Note 11
).
2. Pour gently the previously prepared mixture creating a line in
one of the rails of the IPG strip focusing tray, avoiding the for-
mation of bubbles. Repeat using a clean rail for each sample.
protein and complete to 400
μ
3. Place for each sample, 17 cm-long IPG strips with different
pH range gradient. In this case, pH 3-10 IPG strips with lin-
eal and no lineal gradient were used (
see
Note 20
). Avoid trap-
ping air bubbles between the strip and the sample. The position
of the strip must allow its end marks to coincide with the posi-
tion of the (+, anode) and (−, cathode) electrodes.
4. Repeat the procedure previously described from
steps 4
to
19
in Subheading
3.6.1
, for IPG strip rehydration, electro focus-
ing, second dimension separation and staining.
5. Compare the resolution achieved in the separation between
the evaluated gradients. At this point, it is noticed that the
best separation for extracts obtained from carnation tissues is
obtained when a gradient from 3 to 10 NL is used (Fig.
5
).
