Biology Reference
In-Depth Information
PM
Markers
PM
Markers
STEM
ROOT
pH 3
10
pH 3
10
(KDa)
(KDa)
250
250
150
150
100
75
100
75
50
37
50
37
25
20
25
20
15
10
15
10
Fig. 3
Two-DE of protein extracts obtained using TCA-acetone-phenol protocol from carnation stems and
roots infected with
Fod
using Criterion-Free Stain system Bio-Rad
®
17. Switch off the power supply and remove the gel carefully,
when the electrophoresis is fi nished.
18. Perform the visualization using the Criterion Stain Free Gel
Imaging System and capture the corresponding image.
19. Evaluate the gel image in terms of number of bands, intensity
and resolution (Fig.
3
). The presence of striking indicates the
existence of non-protein contaminants which are affecting the
separation (
see
Note 13
).
The selection of a staining method for obtaining the best sensibil-
ity in bands detection is necessary when a 1-DE in large gels
(20 × 20 cm) analysis for a plant extract is done for the fi rst time.
A comparative analysis using different gel staining methods must
be done, once the protein extraction method had been selected.
3.5 SDS-PAGE
on Large Gels
1. Arrange the complete electrophoresis system for 20 × 20 gels.
In this case, the PROTEAN II XL Bio-Rad system was used
(
see
Note 6
).
2. Prepare the separation gel (
see
Note 14
).
3. Prepare the concentration gel (
see
Note 15
).
4. Place the comb into the gel cassete assembly and when ready
to pour the gel, transfer the gel solution between the glass
plates using a pipette.
5. Once the gel polymerization has fi nished place the gel cassete
assembly into the electrophoresis chamber according to the
manufacturer's instructions.
6. Gently remove the comb out of the gel and add enough run-
ning buffer into the electrophoresis chamber. Using a pipette,
wash the wells with the same solution.
