Proteomic Analysis of Proteins Secreted by Streptococcus pyogenes - Bacterial Pathogenesis: Methods and Protocols - page 19

2. Cut a sample application piece in half and place each half in a separate tube. Use
half of a sample application piece for each gel being run.
3. Add 5 μL of the SDS-PAGE molecular weight standard solution to a sample
application piece. Add two drops of molten agarose sealing solution to the sample
application piece ( see Note 27 ).
3.4.2. Equilibration of IPG strips
1. Remove strips from freezer.
2. Add 5 mL of the equilibration buffer containing 100 m M DTT to each tube and
place on a rocking platform for 15 min.
3.4.3. Running Second Dimension
1. Remove the strip from the screw cap tube and dip it in 1× SDS electrophoresis
buffer.
2. Position the strip between the gel plates with the plastic backing against one of
the glass plates.
3. With a thin plastic ruler, or similar instrument, gently push the IPG strip down
so the entire lower edge of the IPG strip is in contact with the top surface of the
slab gel ( see Note 28 ).
4. Apply a sample application piece containing molecular weight markers at the far
end of the strip where there is no IPG gel. Make sure the sample application piece
touches the top surface of the second dimension gel.
5. Gently overlay the IPG strip and sample application piece with agarose sealing
solution. Use care not to introduce bubbles while applying the agarose sealing
solution.
6. Place gels in the Mini-PROTEAN 3 Dodeca Cell electrophoresis chamber.
7. Set the power supply for the following parameters:
a. 50 V for 15 min.
b. 100 V for 2-3 h.
8. Monitor the migration of the bromophenol blue dye and turn off the power supply
when the dye is a few millimeters above the bottom of the gel.
9. Remove gel cassettes from chamber, and using a plastic wedge, carefully open
the cassette ( see Note 29 ).
3.5. Protein Detection, Quantitation, and Analysis
1. Place the gels in fixing solution (10% methanol, 6% glacial acetic acid) for
30 min.
2. Pour off fixing solution.
3. Add 30 mL of SYPRO Ruby stain. Place the gels on a rocking platform and stain
overnight in a foil covered or lightproof plastic container ( see Note 30 ).
4. The next day, pour off the SYPRO Ruby stain.