Biology Reference
In-Depth Information
11. Fill the tube with 0.1 M ammonium acetate in 100 % (v/v)
methanol, mix and allow precipitating overnight at −20 °C.
12. Centrifuge at 16,000 × g for 5 min at 4 °C and discard the
supernatant.
13. Wash the pellet with 100 % (v/v) methanol and mix well by
vortexing.
14. Centrifuge at 16,000 × g for 5 min at 4 °C and remove the
supernatant.
15. Wash the pellet with 80 % (v/v) acetone and mix well by
vortexing.
16. Centrifuge at 16,000 × g for 5 min at 4 °C and discard the
supernatant.
17. Air-dry the pellet to remove acetone.
18. Dissolve the pellet in 200
μ
L of the solubilization solution,
and shake for 4 h at 4 °C.
19. Quantify proteins using the Bradford method [ 12 ].
20. Store the extract at −20 °C for further analysis.
Selection of the best extraction procedure will depend on pro-
tein yield, and number of bands or spots visualized and resolved in
1-DE or 2-DE gels. Protein yield was determined as
g of BSA
equivalents/mg of lyophilized plant material ( see Notes 4 and 5 ).
μ
3.3 One-Dimensional
Electrophoresis
(SDS-PAGE) on Small
Gels
Arrange the complete electrophoresis system for small gels ( see
Note 6 ). In this case, the Criterion Stain Free Gel system was used.
1. Remove the comb from the gels carefully and insert the system
in the electrophoresis chamber according to the manufactur-
er's instructions. For instance, pre-cast polyacrylamide gels
(4-20 % gradient) can be used ( see Note 7 ).
2. Add enough electrophoresis buffer to the electrophoresis
chamber to the top of the gel and wash the wells with the same
solution.
3. Prepare the samples using the different extracts obtained from
different methods. Take a volume containing 15
μ
g of protein
and adjust with water to a volume of 10
μ
L. Add 5
μ
L of load-
ing buffer and mix by vortexing.
4. Simultaneously, prepare the molecular weight markers
(Precision Plus Protein Standards).
5. Heat the sample and markers for 5 min at 100 °C, and load
them in each of the wells carefully.
6. Close the electrophoresis chamber and set at 150 V for run-
ning. Unplug the power supply and remove the gel carefully
when the electrophoresis is fi nished.
Search WWH ::




Custom Search