Proteomic Analysis of Proteins Secreted by Streptococcus pyogenes - Bacterial Pathogenesis: Methods and Protocols - page 18

3. Centrifuge samples at 25,000 × g for 10 min at 4 °C. Transfer the supernatant
fluid to a clean microcentrifuge tube.
4. Slowly pipet 125 μL ( see Note 19 ) of the sample to the center of a 7-cm ceramic
strip holder ( see Note 20 ). Remove any bubbles.
5. Remove the protective plastic cover from the IPG strip. Position the IPG strip
with the gel side down and the anodic end of the strip directed toward the anodic
end of the strip holder. Lower the strip into the sample starting with the anodic
end ( see Note 21 ). Finally, lower the cathodic end of the IPG strip into the
channel, making sure that the gel contacts the strip holder electrodes at each
end. Be careful not to trap bubbles under the IPG strip.
6. To minimize evaporation and urea crystallization, apply IPG strip cover fluid.
7. Place the cover on the strip holder and place the ceramic holder on the IPGphor.
8. Rehydrate and focus the strips according to the following parameters:
a. Rehydration for 12 h at 20 °C ( see Note 22 ).
b. Isoelectric focusing (IEF) parameters: 20 °C, 50 μA per strip.
c. Step 1: 500 V for 500 V/h (step and hold).
d. Step 2: 1000 V for 1000 V/h (step and hold).
e. Step 3: 8000 V for 32,000 V/h (step and hold).
9. Use forceps to remove strip from the ceramic strip holder and place IPG strips
into separate 15-mL polypropylene centrifuge tubes with the support film toward
the tube wall ( see Note 23 ).
10. Proceed to Subheading 3.4.2 or store the IPG strips at -80ºC.
3.3. Casting Polyacrylamide Gels
1. Assemble the Mini-PROTEAN 3 gel casting chamber ( see Note 24 ).
2. Prepare a 12% acrylamide gel solution. For six gels (8 × 7.3 × 0.001 cm), mix
13.5 mL of 40% acrylamide/bis-acrylamide stock solution, 18.6 mL of deionized
water, 12 mL of 1.5 M Tris-HCl (pH 8.8), and 0.45 mL of 10% SDS. Mix well.
Just before pouring gels, add 0.45 mL of 10% ammonium persulfate and 12.6 μL
TEMED and mix well ( see Note 25 ).
3. Immediately pour the acrylamide solution into the casting chamber, filling it until
the solution is approximately 3 mm from the top of the short plate.
4. Gently overlay each gel with water-saturated butanol.
5. Allow the gels to polymerize 45-60 min.
6. Just before electrophoresis, disassemble the casting chamber.
7. Rinse the gels with distilled water to remove any butanol.
3.4. Second-Dimension Gel Electrophoresis
3.4.1. Preparation for Electrophoresis
1. Place 3.4-4.4 L of 1× SDS electrophoresis buffer in the Mini-PROTEAN 3
Dodeca Cell chamber and pre-cool the buffer by setting the MultiTemp III
Thermostatic Circulator to 10 °C ( see Note 26 ).