Comparative Analyses of Protein Complexes by Blue Native DIGE - Difference Gel Electrophoresis (DIGE): Methods and Protocols - page 152

procedure. Using the CyDye™ fl uorophores, gels should be
scanned at 50-100 mm resolution at the appropriate excitation
wavelengths (488 nm for Cy2™, 532 nm for Cy3™, and 633 nm
for Cy5™). Digital gel images can be visualized using the
ImageQuant analysis software (GE Healthcare). Quantifi cation of
relative differences of individual protein abundances can be carried
out using specifi c software such as Delta 2D (Decodon, Greifswald,
Germany) or DeCyder™ (GE Healthcare).
4. Notes
1. Digitonin is necessary for the solubilization of membrane-
bound protein complexes of cellular or organellar fractions.
Other nonionic detergents (e.g., dodecylmaltoside and Triton
X-100) can be used but should be applied in the presence of
modifi ed buffer systems; see Wittig et al. ( 12 ).
2. The pH value of the buffer must be >9. Normally, it is at about
pH 10 without adjustment. Therefore, the buffer can be
directly used.
3. Always use CyDyes from the same reaction kit diluted with
DMF of one batch to assure comparative labeling conditions.
Consume diluted CyDyes within 3 months.
4. If very large protein complexes (>3 MDa) have to be resolved,
the acrylamide gradient gel of the BN gel can be substituted by
a 2.5% agarose gel prepared in BN gel buffer ( 13 ).
5. The resulting detergent-to-protein ratio is 5:1 (w/w).
6. A pH value of about 8.5 is a prerequisite for effi cient labeling.
The pH value of the protein solution can be easily controlled
using pH test strips.
7. Additionally, a third fraction containing a mixture of both pro-
tein fractions (50 mg protein each) can be labeled with a third
CyDye as internal standard.
8. Coomassie Blue might quench the fl uorescence signal of the
CyDyes. If the cathode buffer is substituted by the same buffer
lacking Coomassie Blue after half of the electrophoresis run,
the background of the resulting BN gel is much clearer.
Alternatively, native gel electrophoresis can be performed fol-
lowing the “clear native” protocol of Wittig et al. ( 14 ).
9. With respect to the Tricine-SDS PAGE system for the second
gel dimension, Schägger and von Jagow ( 11 ) originally pro-
posed a two-step separation gel consisting of a large 16% phase
and a smaller 10% phase (called “spacer gel”). The advantage
of this slightly more complicated gel system is a better resolution