2D DIGE Saturation Labeling for Minute Sample Amounts - Difference Gel Electrophoresis (DIGE): Methods and Protocols - page 111

result in incomplete labeling, leading to a different image as
compared to the analytical gel and probably impairing a cor-
rect spot assignment between analytical and preparative gels.
18. Keratin contaminations cause problems in mass spectrometry
for two reasons: (1) in human samples, exogenously intro-
duced human keratin cannot be discriminated from endoge-
nous keratin potentially contained in the sample; (2) tryptic
peptides derived from exogenous keratin are potent competi-
tors in peptide ionization. The resulting ion suppression may
impair the identifi cation of weak spots. Beware that keratin
contaminations in solvents or solutions cannot be rendered
innocuous by autoclaving. Use carefully prepared fresh solu-
tions for gel casting, gel staining, etc., in keratin-free labware.
19. As a consequence of labeling cysteine residues to completion,
spot intensity is determined not only by the amount of protein
contained in the spot but in addition by the number of cysteine
residues per molecule. Spot intensities obtained by staining
procedures, e.g., Coomassie or Sypro Ruby®, do not at all
refl ect the cysteine content but are predominantly dependent
on the protein amount. Intense spots in the Cy3 image may
therefore appear as weak spots in the Coomassie gel and vice
versa, and some spots visible in the Coomassie gel may not be
visible in the Cy3 gel when no cysteine residue is present in the
protein. Therefore, the CyDye image may differ considerably
from the Coomassie image of the same gel. A spot assignment
in the preparative gel should therefore always be based on the
comparison of analytical and preparative CyDye images.
20. Take into account that after Coomassie staining, a further Cy3
readout is impaired by Coomassie-derived fl uorescence signals
and absorption effects.
Acknowledgments
The authors would like to extend their appreciation to the Deutsche
Forschungsgemeinschaft (DFG) for ongoing research grants within
the research unit FOR 1041, previous grants within FOR 478 and
GRK 1029, the Federal Ministry for Education and Research
(BMBF) for ongoing research grants within PHANOMICS,
FUGATO and FUGATO-plus (REMEDY), the European Union
for a research grant within the Plurisys consortium, and the
European Science Foundation (ESF) for funding of the “Stressfl ea”
consortium. The authors wish to acknowledge their appreciation
to Patrick Bolbrinker, Myriam Demant, Daniela Deutsch, and Julia
Korte for critical reading of the manuscript and to Miwako Kösters
for technical assistance.