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The Electrophoresis Laboratory at Geneva University Hospital
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| Two-dimensional polyacrylamide gel electrophoresis (2D PAGE) is one of the most powerful techniques for the study of protein expression and their post-translational modifications. However, the separation of low copy number proteins in amounts sufficient for post-separation analysis continues to present a challenge for 2-D techniques. Improvements in shortening the focusing time, increasing the loading capacity and enhancing resolution is still needed. |
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A. IPG Gel Strips Rehydration and Sample Application
1. A Methacrylate reswelling chamber was built to accommodate 10 IPG strips in separate grooves. Each groove is 10 mm deep, 4.0 mm wide and 205 mm long (see Hint #1).
2. Up to 15 mg of protein can be solubilized with 500 μl of Sample Rehydration Solution
3. Pipet the entire protein samples into the grooves of the chamber.
4. Position narrow (1 pH unit) or wide range IPG strips in the chamber such that the gel of the strip is in contact with the sample (upside down).
5. Cover the gel and the sample with 3 ml of low viscosity paraffin oil to avoid evaporation.
6. Leave strips at room temperature for six hours to overnight.
7. Remove the rehydrated IPG gel strips carrying the protein sample from the grooves with tweezers and rinse with water.
8. Position the gel strips on the strip tray as described by the manufacturer.
B. Running Conditions
1. Increase the voltage linearly from 300 to 3,500 V during 3 hours.
2. Run gel for an additional 3 hours at 3,500 V.
3. Increase the voltage to 5,000 for the remaining period.
4. Carry out focusing for a total of 100 kVolthours (kVh).
5. After running, strips can be frozen (-20°C) for several weeks (remove oil first), or used immediately for the second dimension.
C. IPG Gel Strips Equilibration (see Hint #2)
1. Equilibrate IPG strips in 3 ml of Equilibration Buffer per groove in the reswelling chamber for 12 min.
2. Aspirate the solution.
3. Block free sulfhydryls by adding Sulfhydryl Blocking Buffer.
4. Incubate for 5 min.
5. Continue with running second dimension (see Protocol on SDS-PAGE For Second Dimension of Immobilized pH Gradient Strips).
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| Sulfhydryl Blocking Buffer |
| 30% (v/v) Glycerol
trace amounts Bromophenol Blue
50 mM Tris-HCl, pH 6.8
2% (w/v) SDS
2.5% (w/v) Iodoacetamide
6 M Urea
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| Equilibration Buffer |
| 2% (w/v) DTE
30% (v/v) Glycerol
50 mM Tris-HCl, pH 6.8
2% (w/v) SDS
6 M Urea
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| Sample Rehydration Solution |
| 8 M Urea
trace amounts Bromophenol Blue
0.8% (w/v) Resolyte, pH 4.0 to 8.0 depending on your pH range
65 mM Dithioerythritol (DTE)
4% (w/v) CHAPS
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Glycerol
Dithioerythritol
CHAPS
Urea
Resolyte
Tris
IodoacetamideParaffin Oil, low viscosity
Bromophenol Blue
SDS
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1. Four leveling feet and a spirit level are included in the chamber. This chamber can be easily constructed in workshops.
2. After the first dimension run, the strips are equilibrated in order to resolubilize the proteins and to reduce disulfide bonds.
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