Proteomic Analysis of Proteins Secreted by Streptococcus pyogenes - Bacterial Pathogenesis: Methods and Protocols - page 20

5. Add 30 mL of destain (10% methanol, 6% glacial acetic acid) and place the gels
on a rocking platform. Allow the gels to destain for 4-24 h ( see Note 31 ).
6. Pour off the destain.
7. Add distilled water to the gels ( see Note 32 ).
8. Image the gel with a Typhoon 9410 instrument using the 610BP 30 filter and 457
(blue) laser.
4. Notes
1. Use TCA and acetone in a ventilated fume hood and wear suitable protective
gear and clothing.
2. Chemically defined media are preferred because they lack the peptides present
in complex media. If complex media are used, adjustments must be made
empirically in the amount of protein to load because the peptides will alter
protein determinations.
3. Store at 4 °C and prepare fresh weekly.
4. It is safer to purchase liquid solutions of acrylamide:bis-acrylamide rather than
purchasing the powdered form of the reagents because mixing the dry reagents
may create dust that is harmful if inhaled.
5. To prevent gels from overheating during electrophoresis, one needs a circulating
cooler when running the Mini-PROTEAN 3 Dodeca.
6. Heating solutions containing urea can cause protein carbamylation (9) .
7. Up to 100 m M DTT can be added, if desired.
8. SDS electrophoresis buffer can be made as a 10× solution (250 m M Trizma base,
1.92 M glycine, 1.0% (w/v) SDS) and diluted to 1× as needed. Our laboratory
makes a carboy of 10 L of 10× buffer and we store it at room temperature.
9. The addition of just a few grains of bromophenol blue is sufficient. This will
provide a dye marker to monitor electrophoresis.
10. Relatively few proteins are detected in supernatant fluids of exponential phase
cultures.
11. The CSPs can now be stored at -80 °C for up to 1 week. -80 °C storage is for
convenience only and does not improve protein recovery.
12. Supernatants should be ice-cold but not frozen.
13. Solution may become cloudy.
14. If sample volume is more than 3 mL, dispense 750 μL into the appropriate
amount of 1.5-mL tubes until the entire sample has been divided.
15. Adding 10 μL of the 10× DNase buffer assumes there is a final volume of
1.0 mL. If the volume is different, add the appropriate amount of 10× DNase
buffer.
16. Resuspension volume will range from 100 to 350 μL depending on the size of
the pellet. Start off with a small volume and vortex well. Continue adding SR
solution until all of the pellet is resuspended. It may be necessary to rock the
samples overnight to get the best resuspension of the precipitated protein. Using
the smallest volume of SR solution necessary to dissolve the pellet will give a