Protein Extraction and Gel-Based Separation Methods to Analyze Responses to Pathogens in Carnation ( Dianthus caryophyllus L) - Plant Proteomics: Methods and Protocols - page 558

7. Prepare the samples using a sample volume containing 100 μ g

of protein and add water to a volume of 20 μ L. At this point,

L of loading buffer and mix by vortexing.
8. Simultaneously, prepare the molecular weight markers (SDS-
PAGE Broad Range markers). Heat the samples and markers for
5 min at 100 °C and load them in each of the wells carefully.

add 10 μ

9. Close the electrophoresis chamber and set at 100 V for run-
ning overnight at 4 °C. Unplug the power supply and remove
the gel carefully when the electrophoresis is fi nished.
10. Stain the gels according to the protocols available in your
laboratory. In this case two protocols were evaluated: SYPRO
Ruby staining and SYPRO Ruby-Coomassie colloidal staining
( see Note 16 ).

1. Wash the gel for 30 min with the fi xing solution.
2. Gently remove the fi xing solution and add SYPRO ® Ruby Bio-
Rad. Shake overnight in the dark (16-18 h).
3. Wash the gel with the fi xing solution for 30-60 min.
4. Wash the gel using lots of water.
5. Capture the image in the Fluorescence Scanner device (FX Pro
Plus Multiimager, Bio-Rad).

3.5.1 SYPRO Ruby
Staining

1. Remove the SYPRO Ruby from the gel. In order to do so,
wash it 3 to 4 h with the gel washing solution.
2. Wash with ddH 2 O for 30 min.
3. Add Coomassie colloidal solution and keep the gel for 60 h in
continuously shaking.
4. Wash the gel using destaining solution A for 3 min.
5. Wash using destaining solution B for 1 min.
6. Wash using destaining solution C at least 24 h.
7. Capture the image in the calibrated densitometer (GS-800
Calibrated Densitometer, Bio-Rad).

3.5.2 SYPRO Ruby-
Coomassie Colloidal
Staining

Compare the number of bands for each of the tested treatments
using the Quantity One ® software from Bio-Rad.

3.5.3 Gel Image Analysis

1. Load the images in the Quantity One ® Bio-Rad software
format. Images must share the same format and resolution.
2. Perform the automatic detection of bands on the different gels
under the same sensitivity conditions. Verify that each band
detected corresponds to one band properly.
3. Compare the number of detected bands and selected the more
sensitive method ( see Note 17 ) (Fig. 4 ).