Proteomic Analysis to Investigate Regulatory Networks in Staphylococcus aureus - Bacterial Pathogenesis: Methods and Protocols - page 34

will need to be adjusted before labeling. The pH can be increased to pH 8.5 by
the careful addition of 50 m M NaOH.
3. For labeling experiments, prepare 400 μ M CyDye working solution by adding one
volume of CyDye stock solution to 1.5 volumes of high-grade DMF ( see Note 4 ).
4. It is recommended that 50 μg of protein is labeled with 400 pmol of CyDye. If
labeling more than 50 μg of protein, the same fluorochrome-to-protein ratio must
be used.
5. Add a volume of sample equivalent to 50 μg of protein and 1 μL of diluted CyDye
to a microfuge tube, mix, and centrifuge briefly. Afterward, incubate the sample
on ice for 30 min in the dark.
6. Stop the labeling reaction by adding 1 μL of 10 m M lysine to the sample that
has incubated on ice for 10 min in the dark. Labeled protein extracts can now be
stored at -80 °C.
7. The protein samples that are going to be separated on the same gel should be
pooled now.
8. Follow the protocol for rehydration of IPG strips ( see Subheading 3.4.1. ).
3.4. 2D Gel Electrophoresis
3.4.1. Isoelectric Focusing
1. Make up protein samples [80-100 μg of radioactively labeled proteins, 3 × 50 μg
of DIGE-labeled proteins ( see Note 5 ), or 350-600 μg of unlabeled proteins
for Colloidal Coomassie staining) ( see Note 6 )] to 360 μL with 8 M urea/2 M
thiourea. Subsequently, add 40 μL of 10× rehydration buffer and mix the solution
by shaking at room temperature for 30 min. Centrifuge the rehydration mix for
5 min at 21,000 × g (20 °C) to remove insoluble proteins.
2. Dispense equally the supernatant in one slot of the rehydration chamber. Position
the IPG strip with the gel side down and lower the strip onto the solution. To
help coat the entire IPG strip, gently lift and lower the strip and slide it back and
forth along the surface of the solution. Avoid large bubbles under the IPG strip.
Rehydration occurs overnight for at least 15 h (no longer than 24 h).
3. Perform IEF with the Multiphor II unit. The dry-strip aligner and the electrodes
should be cleaned with soft tissues and all cables should be connected. Set the
temperature of the thermostatic circulator to 20 °C. Place two 110-cm electrode
strips on a clean flat surface and soak with distilled water. Remove excess water
by blotting with filter paper ( see Note 7 ).
4. To remove rehydrated IPG strips from the slots, grab the ends of the strip with
tweezers and lift it out of the tray. Any contact with the gel surface should be
avoided. Position the strips in the dry-strip aligner in adjacent grooves and align
all strips so that the anodic gel edges are lined up. Place the electrode strips across
the cathodic and anodic ends of the aligned IPG strips. The electrode strips must
at least partially contact the gel surface of each IPG strip. When each electrode
is aligned with the electrode strip, press down on each to contact the electrode
strips. Cover the IPG strips with mineral oil.