Biology Reference
In-Depth Information
2.3. Two-Dimensional Polyacrylamide Gel Electrophoresis
2.3.1. First Dimension
1. Rehydration solution (10×): 100 mg of CHAPS (Roth, Karlsruhe, Germany) and
30 mg of DTT (MP Biomedicals, Illkirch, France) in 8 M urea/2 M thiourea
solution ( see step 16 , Subheading 2.1. ). Subsequently, add 52 μL Pharmalyte
(pH 3-10) (GE-Healthcare). Store aliquots at -20 °C.
2. 18-cm IPG strips: linear pH range of 4-7 for cytoplasmic proteins and 3-10 for
extracellular proteins (GE-Healthcare).
3. Rehydration chamber.
4. Multiphor II unit (GE-Healthcare).
5. Electrode strips (GE-Healthcare).
2.3.2. Second Dimension
1. 1.5 M Tris-HCl, pH 8.8 (adjusted at 12 °C). Store at room temperature ( see Note 1 ).
2. 10% (w/v) sodium dodecyl sulfate (SDS) in deionized water. Store at room
temperature.
3. 10% (w/v) ammonium persulfate (APS) dissolved in deionized water. Store at
4 °C no longer than 1 week.
4. 0.5 M Tris-HCl, pH 6.8 (adjusted at 12 °C). Store at room temperature ( see Note 1 ).
5. Upper buffer (4×): 0.4% (w/v) SDS dissolved in 0.5 M Tris-HCl, pH 6.8.
6. Equilibration solution (100 mL): Dissolve 36 g of urea in a mixture of 10 mL
of 0.5 M Tris-HCl (pH 6.8), 30 mL of glycerol, and 40 mL of 10% SDS. For
solution A, add 175 mg of DTT (MP Biomedicals) to 50 mL equlibration solution.
For solution B, add 2.25 g of iodoacetamide (toxic!) and a trace of bromphenol
blue to 50 mL equilibration solution.
7. Running buffer (10×): Dissolve 150 g of Tris base, 720 g of glycine, and 50 g
of SDS in deionized water. When all components are completely in solution, add
deionized water to 5 L.
8. Protean® plus Dodeca™ cell gel system, power supply, glass plates (Biorad-
Laborities, Hercules, USA).
2.4. Protein Detection
1. The Coomassie staining solution is prepared as follows: add 40 mL of 85%
o -phosphoric acid, 40 g of (NH 4 ) 2 SO 4 , and 0.48 g of Coomassie Blue G-250 to
360 mL deionized water. Stir solution and add 100 mL methanol.
2. Fixing solution for silver staining: 50% (v/v) ethanol and 12 % (v/v) acetic acid.
Prior to use, add 500 μL of 37% formaldehyde to 1 L fixing solution.
3. Washing solution: 50% (v/v) ethanol in deionized water.
4. Sodium thiosulfate solution: Add 0.2 g of Na 2 S 2 O 3 ·5H 2 O to 1 L deionized water
and dissolve by stirring.
5. Silver nitrate solution: Add2gofAgNO 3 to 1 L deionized water and dissolve
by stirring. Prior to use, add 750 μL of 37% formaldehyde to 1 L silver nitrate
solution.
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