Proteomic Analysis to Investigate Regulatory Networks in Staphylococcus aureus - Bacterial Pathogenesis: Methods and Protocols - page 36

6. Perform electrophoresis at constant power (2 W per gel) at 12 °C. A constant
temperature during electrophoresis is very important for gel-to-gel reproducibility.
When starting electrophoresis, the buffer temperature should be at 12 °C.
Therefore, cooling has to be started before preparing the gels ( see step 1 , this
Subheading ).
7. After electrophoresis, remove gels from glass plates in preparation for staining.
Each gel should be marked to identify the acidic end of the first dimension
preparation.
3.5. Protein Detection
3.5.1. Colloidal Coomassie Staining (12)
1. Add 250 mL of Coomassie staining solution to each gel and incubate gels for
24 h on a stirrer.
2. After removing Coomassie staining solution, rinse the gels with deionized water
several times.
3. Seal gels in foil after staining.
4. Scan gels with a Scanner X finity ultra (Quato Graphic) in transmission mode at
a resolution of 200 dpi.
5. Store gels at 4 °C.
3.5.2. Silver Staining (13)
1. Treat the resulting 2D gels with fixing solution for silver staining (200-250 mL
per gel) for 1-2 h.
2. Wash gels three times with 200-250 mL of 50% (v/v) ethanol for 20 min and
pretreat with 200-250 mL of sodium thiosulfate solution for 1 min.
3. Rinse the gels three times with deionized water for 20 s.
4. Incubate gels with 200-250 mL of silver nitrate solution for 20 min.
5. After removing the silver nitrate solution, rinse the gels twice with deionized
water for 20 s.
6. For developing, incubate gels in 200-250 mL of potassium carbonate solution for
2-10 min. Stop the developing reaction by incubating the gels with 200-250 mL
of 1% (w/v) glycine for 20 s, rinsing with deionized water for 20 s and stopping
again with 200-250 mL of 1% (w/v) glycine for 20 min.
7. Wash gels two times with deionized water for 20 min and seal in foil.
8. Scan gels with a Scanner X finity ultra (Quato Graphic) in transmission mode at
a resolution of 200 dpi.
9. Store gels at 4 °C.
3.5.3. Detection of DIGE-Labeled Proteins
Cy2, Cy3, and Cy5 can be detected using a Typhoon laser scanner (9200,
9210, 9400, or 9410). The unfixed gels are scanned according to the Ettan Dige
User manual (GE-Healthcare).