Biology Reference
In-Depth Information
5. Sample buffer (2×): 7 M urea, 2 M thiourea, 4% CHAPS,
130 mM DTT, 30 mM Tris-HCl (pH 8.5), trace BPB.
6. Reswelling tray for 24-cm strip.
7. Multiphor™ II and Immobiline Dry Strip cover fl uid (GE
Healthcare).
8. Power supply: EPS 3501 XL power supply (GE Healthcare).
9. Thermostatic circulator: MultiTemp III thermostatic circula-
tor (GE Healthcare).
10. Carrier ampholyte mixtures: IPG buffer for pH 3.5-4.5, pH
4.0-5.0, pH 4.5-5.5, pH 5.0-6.0, pH 5.5-6.7, pH 7-11, pH
3-10 NL, pH 4-7 (GE Healthcare).
11. Gradient former: Model 395 (Bio-Rad, Milan, Italy).
12. SDS PAGE gel cast: Ettan DALTtwelve Electrophoresis System
(GE Healthcare).
13. Ettan DALT low fl uorescence (LF) glass plate set (26 × 20 cm)
(GE Healthcare).
14. Ettan DALT glass plate set (26 × 20 cm) (GE Healthcare).
15. Tris-HCl buffer (5×): 227 g Tris in 1 L of distilled water
(adjusted to pH 8.8 with concentrated HCl).
16. SDS buffer (5×): 15 g Tris, 72 g glycine, and 5 g sodium dode-
cyl sulfate (SDS) in 1 L of distilled water (pH 8.8).
17. Acrylamide stock solution: Acrylamide/Bis-acrylamide 37:5.1,
40% (w/v) solution (AMRESCO, Solon, OH, USA).
18. Equilibration buffer: 180 g urea, 10 g SDS, 100 mL of 5× Tris-
HCl buffer, 200 mL of 50% (v/v) glycerol, 31.25 mL of acrylam-
ide stock solution, 5 mM tributylphosphine (TBP) (see Note 2).
19. Gel solution for making 14 gels (26 × 20 cm, 1-mm spacer,
9-16% gradient): Heavy solution (93.4 mL of 5× Tris-HCl
buffer, 199 mL of 40% acrylamide stock solution, 175 mL of
50% glycerol, 1 mL of 10% (w/v) ammonium persulfate (APS),
and 100
L TEMED); light solution (93.4 mL of 5× Tris-HCl
buffer, 105 mL of 40% acrylamide stock solution, 1 mL of 10%
(w/v) APS, 100
μ
L TEMED, and 269 mL distilled water).
20. Fixing solution: 40% (v/v) methanol and 5% (v/v) phosphoric
acid in distilled water.
21. Coomassie Brilliant Blue G-250 staining solution: 17% (w/v)
ammonium sulfate, 3% (v/v) phosphoric acid, 34% (v/v)
methanol, and 0.1% (w/v) Coomassie Brilliant Blue G-250 in
distilled water.
22. Preparative gel scanner: GS710 model (Bio-Rad), 100-
μ
μ
m
high-resolution unit.
23. 2D DIGE gel scanner: Typhoon 9400 imager (GE Healthcare).
24. Image preprocessor: ImageQuant V2005 (GE Healthcare).
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