Biology Reference
In-Depth Information
6.
Combine all three CyDye-labeled protein samples in one
reagent tube.
7.
Add the remaining volume of rehydration buffer containing
DTT and IPG buffer to the fi nal volume required for IEF
separation.
8.
Perform 2D DIGE in the dark. For the second dimension SDS
PAGE, the Laemmli or the Schägger protocol can be applied
( 18, 21 ).
9.
After fi nishing the electrophoretic separation, the gel is imme-
diately scanned using a fl uorescence scanner (Typhoon
Fluorescence Scanner; GE Healthcare, Munich, Germany).
Keep the gel at 4°C and in the dark before starting the scanning
procedure. Using the CyDye fl uorophores, gels have to be
scanned at 50-100-
m 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, Munich,
Germany) (Fig. 1 ). Quantifi cation of relative differences of indi-
vidual protein abundances can be carried out using specifi c
software (e.g., Delta 2-D (Decodon, Greifswald, Germany) or
DeCyder™ (GE Healthcare, Munich, Germany)).
μ
Fig. 1. Two-dimensional DIGE analysis of total protein extracts from zygotic embryos (ZE)
and somatic embryos (SE) from Cyclamen persicum. Proteins of each tissue were
extracted as described in this chapter. Protein fractions of ZE and SE were loaded onto
one gel, 100 μ g of each fraction. The ZE protein fraction was labeled with Cy3™ ( red ), the
SE protein fraction with Cy5™ ( green ). Spots with similar abundance in both tissues are
colored yellow. An internal standard was not included.
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