2D DIGE for the Analysis of RAMOS Cells Subproteomes - Difference Gel Electrophoresis (DIGE): Methods and Protocols - page 242

2.5. Isoelectric
Focusing

1. Rehydration buffer: 7 M urea, 2 M thiourea, and 4% (w/v)
CHAPS, stored in aliquots at −20°C. For use, add carrier
ampholytes and DeStreak and/or dithiothreitol (DTT) (see
Notes 8-10).
2. Immobiline DryStrips (GE Healthcare).
3. Immobiline reswelling tray (GE Healthcare).
4. Cover fl uid (GE Healthcare).
5. IPGphor cup-loading strip holder (GE Healthcare).
6. Electrode pads (GE Healthcare).
7. Ettan IPGphor IEF unit (GE Healthcare).

1. Equilibration solution: 50 mM Tris-HCl, pH 8.8, 6 M urea,
30% (v/v) glycerol, 2% (w/v) SDS, and trace bromophenol
blue. Store in aliquots at −20°C.
2. Reduction solution: equilibration solution containing 1% DTT.
3. Alkylation solution: equilibration solution containing 4% iodo-
acetamide (see Note 11).

2.6. Equilibration
Solutions

2.7. SDS-
Polyacrylamide
Gel Solutions

1. 0.3% (w/v) agarose in running buffer 1×. Store at 4°C.
2. Water-saturated butan-2-ol (see Note 12).
3. SDS electrophoresis buffer:
1× = (25 mM Tris-HCl, 192 mM glycine, 0.1% (w/v) SDS) for
lower tank.
2× = (25 mM Tris-HCl, 192 mM glycine, 0.2% (w/v) SDS) for
top tank.
Store at room temperature (RT) (see Note 13).

4. Monomer stock solution: 30% (w/v) acrylamide and 0.8%
(w/v) methylenebisacrylamide. Electrophoresis purity reagent
Bio-Rad (see Note 14). Store at 4°C in dark.
5. Resolving gel buffer: 1.5 M Tris-HCl and pH 8.8. For 1 L of
solution, add approximately 33 mL of concentrated HCl
(12 N), then make up to 1 L with water. Store at 4°C.
6. 10% (w/v) SDS (see Note 15).
7. Ammonium persulfate: 10% (w/v) solution in water. Prepare
fresh each time.
8. N , N , N , N ¢-tetramethylethylenediamine (TEMED) (Sigma).
Store at 4°C.
9. 12.5% acrylamide gel solution with 0.2% (w/v) SDS for four
gels: 125 mL of acrylamide solution (30% acrylamide: 0.8%
methylenebisacrylamide), 75 mL of 1.5 M Tris-HCl, pH 8.8,
93.5 mL of Milli-Q water, 6 mL of 10% (w/v) SDS (0.2%
(w/v) fi nal concentration), 3 mL of 10% (w/v) ammonium
persulfate, and 41.5 mL of 100% TEMED.