Biology Reference
In-Depth Information
4. Notes
1. For highly reproducible 2D gels, the pH of these buffers should be adjusted at
a fixed temperature.
2. These protocols are easily adaptable to other organisms. However, you will have
to appropriately modify the culture conditions and the cell disruption protocols.
3. Ensure that at least 10 μL of each protein solution is used. Otherwise, dilute
an appropriate volume of the protein extract prior to measurement. Ensure that
the ratio E
590nm
/E
450nm
is always the same for each protein extract. Protein
extracts in TE or water should be frozen at -20 °C before measuring the protein
concentration.
4. Add the DMF first to the tube followed by the CyDye.
5. When separating DIGE-labeled proteins on 2D protein gels, all steps should be
carried out in the dark.
6. If the volume containing the desired amount of cytoplasmic proteins exceeds
40 μL, the volume should be reduced by using a speed vac. (Caution: this step
can only be applied for protein extracts dissolved in TE buffer!)
7. Important: strips must be damp, not wet!
8. These instructions assume the use of the Protean® plus Dodeca™ cell gel system
(Biorad-Laborities). They are easily adaptable to other gel systems.
9. The total volume of the solution needed depends on the gel sizes, the gel
thickness, and the number of the gels cast.
10. For an optimized scanning process, the gels should be scanned separately by the
three different lasers.
11. Ensure that the gels are completely dried before interrupting the vacuum. The
gel should have lost its softness when you press your finger on it.
12. For quantitation of protein synthesis, ensure that no protein spot is saturated.
Acknowledgments
We are very grateful to Christian Kohler, Anne-Kathrin Ziebandt, Harald
Kusch, Stephan Fuchs, and Robert S. Jack for critical reading the manuscript
and preparing of figures. We thank all co-workers and students for their
excellent work on proteome analyses. Furthermore, we thank the Decodon
GmbH for providing the Delta2D software. This work was supported by grants
from the Bildungsministerium für Bildung und Forschung (031U107A/-207A;
031U213B), the DFG (GK212/3-00), the Land Mecklenburg-Vorpommern, and
the Fonds der Chemischen Industrie to M.H. and S.E.
References
1. Kuroda, M., Ohta, T., Uchiyama, I., Baba, T., Yuzawa, H., Kobayashi, I., Cui, L.,
Oguchi, A., Aoki, K., Nagai, Y., Lian, J., Ito, T., Kanamori, M., Matsumaru, H.,
Maruyama, A., Murakami, H., Hosoyama, A., Mizutani-Ui, Y., Takahashi, N. K.,
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