2D DIGE Saturation Labeling for Minute Sample Amounts - Difference Gel Electrophoresis (DIGE): Methods and Protocols - page 97

performing the optimization experiment. Under these circumstances,
use the most typical sample available instead. If all samples are
already available, prepare the IPS by mixing aliquots comprising
equal protein amounts of all samples integrated in the saturation
DIGE study. In both cases, follow the scheme of Table 1 , using six
5 μ g aliquots of the sample for Cy3 labeling and another six 5 μ g

aliquots for Cy5 labeling:

1. Transfer a 5 μ g protein aliquot into a sterile microfuge tube.

2. Adjust the volume to 9 μ L using the cell lysis buffer.

3. Add the required volume of freshly prepared 2 mM TCEP

solution according to Table 1 to the protein solution of step 1.
Discard any unused material.

4. Mix vigorously by pipetting.

5. Spin down the sample in a microcentrifuge.

6. Repeat steps 4 and 5 to make sure the solution is really homo-

geneous and is located completely at the bottom of the
microfuge tube.

7. Incubate at 37°C for 60 min in the dark.

8. Add the required volume of 2 mM CyDye according to

Table 1 .

9. Mix vigorously by pipetting.

10. Spin down the sample in a microcentrifuge.
11. Repeat steps 9 and 10 to make sure the solution is really homo-
geneous and is located completely at the bottom of the
microfuge tube.
12. Incubate at 37°C for 30 min in the dark.
13. Stop the reaction by adding a volume equal to the reaction
volume of sample buffer II.
14. Mix vigorously by pipetting.
15. Spin down the sample in a microcentrifuge.
16. Samples are ready for 2D PAGE analysis and can be stored
frozen in the dark for up to 1 month, preferably at −70°C.

Before 2D PAGE analysis, combine corresponding Cy3 and
Cy5 samples for each stoichiometric condition. Analyze each
mixture (see Note 9) on the 2D gel electrophoresis equipment
designated for the fi nal study. For isoelectric focusing conditions of
analytical saturation DIGE gels, the protocol given in Table 2 has
been elaborated by the manufacturer for 24-cm pH 3-10 strips,
which should be followed whenever possible.
For the SDS PAGE in the second dimension, make sure to use
low fl uorescent glass plates.
From each of the six gels, generate two fl uorescence readout
images, using a scanner suitable for Cy3 and Cy5 dyes. Laser scanners