Proteomic Analysis to Investigate Regulatory Networks in Staphylococcus aureus - Bacterial Pathogenesis: Methods and Protocols - page 35

5. Perform IEF by using the following voltage profile: step 1—500 V (gradient), 2
mA, 5 W, 2 Vh; step 2—3500 V (gradient), 2 mA, 5 W, 3 kVh; step 3—3500
V, 2 mA, 5W, 23.5 kVh. As IEF proceeds, the current must be checked (low
current is normal for IPG gels). If no current is flowing, check the contact between
electrodes and electrode strips.
6. After IEF, proceed to the second dimension immediately ( see Subheading 3.4.2. )
or store the IPG strips at -20 °C in foil.
3.4.2. SDS-PAGE
Focused proteins are separated according to their molecular weight in 12.5%
acrylamide and 2.6% bis-acrylamide polyacrylamide gels using the Tris-glycine
system ( see Note 8 ).
1. To prepare 12 slab gels, mix 335.3 mL of 40% acrylamide, 179 mL of 2% bis-
acrylamide, 272.54 mL of 1.5 M Tris-HCL (pH 8.8), 11.48 mL of 10% SDS, and
300 mL of deionized water while stirring on a magnetic stirrer. Add 2.8 mL of 10%
APS and 0.55 mL of TEMED to this solution and mix by stirring ( see Note 9 ).
Generation of bubbles should be avoided. Pour gels immediately by filling the gel
cassette about 1 cm below the top of the glass plates. Overlay gels immediately
with a thin layer (1 mL) of water-saturated n-butanol or water to minimize gel
exposure time to oxygen and to create a flat gel surface. Polymerization takes
at least 3 h. Each gel should be inspected and the top of surface of each gel
should be straight and flat. Remove butanol or water and rinse the gel surface
with deionized water. The butanol should be completely removed.
2. Prepare the stacking gel by mixing 10.8 mL of 40% acrylamide, 3.4 mL of 2%
bis-acrylamide, 30 mL of upper buffer (4×), and 74 mL of deionized water.
Afterward, add 0.3 mL of 10% APS and 0.05 mL of TEMED to the solution
while stirring. Use an appropriate amount of the stacking gel solution to quickly
rinse the top of each gel and overlay with a thin layer (1 mL) of water. The
stacking gel should polymerize within 1 h.
3. Prepare equilibration buffers A and B. Place each IPG strips with the gel side up
in one slot of an equilibration chamber and add 4-5 mL of equilibration buffer A
to each slot. Equilibrate the IPGs for at least 15 min with gently shaking. Decant
equilibration solution A and add 4-5 mL of equilibration solution B to each slot.
Equilibrate the IPGs again for at least 15 min while shaking. Decant equilibration
solution B and place the IPG strips on filter paper so that they rest on an edge to
help drain the equilibration solution.
4. Add 1× running buffer to the gel system. Put gels in the gel system filled with
1× running buffer. The running buffer should cover the gels.
5. Place IPG strips between the plates on the surface of the stacking gel by gently
pushing the IPG strip down so that the entire lower edge of the IPG strip is in
contact with the top surface of the stacking gel. No air bubbles should be trapped
between the IPG strips and the stacking gel.