DIGE Analysis of Plant Tissue Proteomes Using a Phenolic Protein Extraction Method - Difference Gel Electrophoresis (DIGE): Methods and Protocols - page 341

7. The second uptake of the phenol phase (step 6) should be
achieved without contamination of the liquid phase. Again,
collection by several small volume steps is recommended.
8. To obtain a very pure pellet, the washing step (step 7) can be
repeated several times.
9. Do not dry the protein pellet for too long under the extractor
hood, because otherwise its resuspension in the lysis buffer
might become diffi cult (1-5 min are usually suffi cient). The
fi nal yield of pellet should be approximately between 5 and
20 mg protein; the protein yield can be determined using a
common protein quantifi cation method.
10. The weight of the dried pellet is close to the total amount of
protein extracted.
11. For an equal protein concentration in the labeling reaction, the
volume for resuspension may vary depending on the protein
amounts of the samples.
12. CyDye labeling most effectively takes place if the pH value of
the lysis buffer is in the range of 8.5. The pH of the sample in lysis
solution can be tested via pH test strips. In case the pH value
is signifi cantly below 8.5, a lysis buffer of higher pH values (e.g.,
pH 9.0) should be used for resuspension of proteins. Always
use CyDyes from one reaction kit diluted with DMF of one
batch to assure comparative labeling conditions. Consume the
CyDyes after diluting them in DMF within 3 months.

Acknowledgments

The authors would like to thank Jennifer Klodmann, Institute for
Plant Genetics, Leibniz Universität Hannover, for critically reading
the manuscript.

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