Biology Reference
In-Depth Information
10. Add 2 mL of hot (75 °C) agarose solution and continue to
slide the strip down onto the surface of the SDS gel until good
contact is achieved. Ensure that no air bubbles are trapped
between the IPG strip and the slab gel surface.
11. Apply the molecular weight markers soaking a fi lter paper pad
with 5
L, let it dry, and apply to the left of the IPG strip.
Allow the agarose to solidify for at least 5 min.
12. Attach the gel sandwich to the upper chamber and place it in the
casting unit. Fill the lower (2-4 L) and upper (450-600 mL)
chamber with running buffer and place the safety lid on the unit.
μ
13. Apply a constant current of 25 mA per gel until the dye reaches
the front of the gel. Then turn off the power supply, discon-
nect the leads, and remove the safety lid.
14. Following electrophoresis pull out the upper buffer chamber
assembly, slide away the spacers, and separate the plates with
the use of a spatula. The gel remains on one of the glass plates.
Invert the plate and position the gel low over the container
with 200 mL of fi xative solution.
Gels are stained according to the method of Neuhoff et al. [
39
].
For each step use a volume of 300 mL per two-dimensional gel and
perform all steps under shaking. Fix the gel for at least 60 min or
overnight in fi xative solution. Decant the fi xer carefully and rinse
the gel three times for 10 min with deionized water to remove the
fi xative solution. Transfer gel to staining solution and leave it over-
night. Rinse the gel repeatedly with warm water (45-55 °C) to
remove residual stain (Fig.
2a
).
3.10 Coomassie
Colloidal Staining
3.11 Image Analysis
Using Progenesis
SameSpots v3.0
(Nonlinear Dynamics)
1. Scan gel images using a transmission-light scanner (Image
Scanner, GE Healthcare) with a resolution of 300 dpi and digi-
talized on 16 bits.
2. Select a good image as a reference with a clear and representa-
tive spot pattern which has a minimum of distortion.
3. Ignore the edges of the gel drawing a mask over these areas.
4. Align each of the images to the chosen reference: Select
between 15 and 20 prominent spots to manually assign and
use the automatic vector tool to add additional vectors.
5. After automatic spot detection and matching, revise manually
the spots with edition tools for correct detection.
6. Establish gel groups according to the experimental design and
normalize spot volume intensity ratios for each spot.
7. List all the spots and their normalized volume.
8. Select the statistically signifi cant differentiated spots across the
experiment based on ANOVA (
p
-value >0.05).
