Biology Reference
In-Depth Information
some mL in the beaker for polymerization control. Add some
2-propanol mL to straighten the level of the gel and remove
unwanted air bubbles. Once polymerization is fi nished, remove
the isopropanol on the top of the gel carefully and wash with lots
of water. Try to remove the residual water excess using fi lter paper.
15. Mix 10.5 mL of ddH
2
O, 3 mL of acrylamide-bisacrylamide
Bio-Rad, 4.5 mL of Tris-HCl pH 8.8 buffer, 180
μ
L of 10 %
(w/v) SDS, and 90
L of 10 % (w/v) APS. Stir the corre-
sponding solution with a magnetic mixer and add 18
μ
μ
L of
TEMED to start the polymerization process.
16. Coomassie colloidal staining shows less sensitivity than SYPRO
Ruby-Coomassie colloidal staining. A higher number of
detected bands with the same amount of protein appeared
with this combined method.
17. SYPRO Ruby and SYPRO Ruby-Coomassie colloidal staining
shows similar sensitivity when compared to the number of
detected bands for the same rail. However, SYPRO Ruby
®
Bio-Rad staining is unstable and can be only visualized with
the system based on fl uorescence detection. By contrast,
SYPRO Ruby-Coomassie colloidal staining has more stability
and allows the evaluation of bands in a visual way. Under these
criteria, the combined SYPRO Ruby
®
Bio-Rad-Coomassie
colloidal staining is selected for carnation extract analysis.
18. The protein amount and the pH gradient on the IPG strips
used in the IEF are determinant factors affecting sensitivity
and resolution in 2-DE. Generally, a greater amount of pro-
tein allows the visualization of a higher number of spots when
low-abundance proteins are present in the extract.
Furthermore, increasing the resolution in the fi rst pI dimen-
sion allows noticing a higher number of spots.
19. A high amount of protein does not necessarily lead to better
results. For some vegetal tissues, when using a high amount of
protein, the threshold of contaminants can be exceeded and
the resolution can be affected during fi rst dimension separa-
tion. Considering, the number of spots and the resolution, the
amount of protein for 2-DE selected is 400
g.
20. No additional gradients were evaluated considering the pres-
ence of spots in the whole range (pH 3-10), according to 2-D
gels electrophoresis on short strips (Subheading
3.4
). Other
separation ranges can be used (for example, pH 5-8) for
increase the resolution in the fi rst dimension separation when
other tissues or species are studied.
21. Other commercial protein IEF markers or pure reference pro-
teins can be used. This is the case of the standard 2D-SDS
PAGE Bio-Rad
®
, which permits the assignment of isoelectric
points in a complex sample when the marker is simultaneously
separated in a 2-D gel under the same conditions.
μ
