2D DIGE for the Analysis of RAMOS Cells Subproteomes - Difference Gel Electrophoresis (DIGE): Methods and Protocols - page 244

4. Centrifuge for 10 min at 15,000 × g at 4°C. Proteins should be
at the interface.
5. Remove carefully the aqueous top layer and discard it.
6. Add 3 volumes of methanol and vortex well.
7. Centrifuge for 5 min at 15,000 × g . Proteins should be seen as
a precipitate.
8. Remove carefully as much liquid as possible, without disturb-
ing the precipitate.
9. Allow the pellet to air-dry for 10-15 min.
10. Store the sample at −20°C until needed.

These steps describe the preparation of a cell lysate compatible
with CyDye DIGE fl uor minimal dye labeling.

3.3. Sample
Solubilization
and Quantifi cation
of Proteins

1. Resuspend the precipitated proteins in a minimal volume of
the lysis buffer and vortex at room temperature for 1 h. If nec-
essary, sonicate intermittently until the precipitate is dissolved
(see Note 16).
2. Centrifuge the cell lysate for 10 min at 15,000 × g in a micro-
centrifuge.
3. Transfer the supernatant which contains the cell lysate to a
labeled tube. Discard the pellet.
4. Check the pH of the cell lysate by spotting 0.2 mL on a pH
indicator strip; it should be pH 8.0-9.0. If the pH is below pH
8.0, it will need to be adjusted before labeling (see Note 17).
5. Determine the quantity of proteins by RC DC protein assay
(see Note 18).

3.4. Preparation
of CyDye DIGE Fluor
Minimal Dyes
for Protein Labeling

1. Take the CyDye DIGE fl uor minimal dye and leave it to thaw
for 5 min at room temperature without opening. This will pre-
vent exposure of the dye to water condensation which may
cause hydrolysis.
2. Dispense a small volume of DMF from its original container
into a microfuge tube. Reconstitute the stock CyDye DIGE
fl uor minimal dye in DMF (see Notes 6 and 7). CyDye fl uor
minimal dye is supplied as a solid and is reconstituted in DMF
to a concentration of 1 nmol/mL.

After reconstitution in DMF, the dye will give a deep color:
Cy2-yellow, Cy3-red, and Cy5-blue. The dye stock solution
(1 nmol/mL) is now prepared. The dye will need to be further
diluted to the working dye solution before being used in the labeling
reaction.