Biology Reference
In-Depth Information
or not lyophilized) can be assayed according to the vegetable
material available.
5. Compare particular results obtained from your system.
According to the evaluation performed in carnation tissues,
TCA-acetone and TCA-acetone-phenol extraction methods
have the best results in terms of protein yield. However, it is
necessary to evaluate the quality of the extracted proteins
using 1-DE. Therefore, this technique can show whether non-
protein contaminant extraction has occurred or not.
6. Clean all the parts of the electrophoresis chamber, glasses,
splitters and combs carefully.
7. For other electrophoretic system, general conditions to per-
form the 1-DE are similar to the ones introduced here, but
following particular specifi cations provided by the particular
electrophoretic system provider. If gels must be prepared, a
discontinuous system with 12 % acrylamide in the separation
gel is recommended.
8. For other electrophoretic systems with no visualization sys-
tem, a Coomassie colloidal staining described later in
Subheading
3.5.2
from
step 2
is recommended.
9. A rapid evaluation can be performed visually by counting
bands on each rail. Likewise, the presence of contaminants in
the extracts can be evaluated by means of visual inspection of
the gels. For this particular case, the TCA-acetone-phenol
method provides the best results in terms of extraction, num-
ber of bands, and extracts cleaning.
10. The fi rst dimension separation using IPG strips is a critical
point during 2-DE, so the presence of non-protein contami-
nants affects its resolution and effi ciency. Therefore, 2-DE
separation is a decisive factor for selecting an extraction
method for proteomic approaches.
11. Final volume varies based on the dimensions of the strips
reported by the manufacturer.
12. Strips can be kept at −20 °C before their use.
13. In this point, the pI range distribution for the separated pro-
teins from a particular extract can be evaluated preliminary.
For extracts obtained from carnation, proteins are distributed
in a 4-10 range principally. According to these results, the
TCA-acetone-phenol method for protein extraction is ade-
quate for proteomic approaches in carnation tissues.
14. Mix 20.2 mL of ddH
2
O, 24 mL of acrylamide-bisacrylamide Bio-
Rad, 15 mL of Tris-HCl pH 8.8 buffer, 600
μ
L of 10 % (w/v)
SDS, and 312
L of 10 % (w/v) APS. Stir the corresponding
solution with a magnetic mixer and add 31
μ
L of TEMED to
start the polymerization process. Quickly, add this solution to the
cassete assembly to 2 cm below the top of the smaller glass. Leave
μ
