Biology Reference
In-Depth Information
7. PROTEAN II xi electrophoresis unit (Bio-Rad).
8. Typhoon 9410 image scanner (GE Healthcare).
9. Orbital shaker/rocker.
10. Sonicator.
2.2. Sample
Preparation
1. ProteoMiner™ small-capacity kit (Bio-Rad) (see Note 1).
2. 2D clean-up kit (GE Healthcare).
3. Coomassie plus protein assay reagent kit (Pierce, Rockford,
IL, USA).
1. CyDye™ DIGE fl uors (minimal dyes): Cy2, Cy3, and Cy5 (GE
Healthcare).
2. 99.8% Anhydrous
N
,
N
-dimethylformamide (DMF) (see Note 2).
3. pH indicator strips.
4. 10 mM
L
-lysine.
5. Cell lysis solution: 7 M urea, 2 M thiourea, 4% CHAPS, 30 mM
Tris, pH being adjusted to 8.5 with HCl.
6. 1 M NaOH.
2.3. DIGE Labeling
1. 40% Acrylamide/bis solution (37.5:1 with 2.6% C) and
N
,
N
,
N
,
N
¢-tetramethylethylenediamine (TEMED) (Bio-Rad).
2. Separating buffer: 1.5 M Tris-HCl, pH 8.8. Store at 4°C.
3. Stacking buffer: 1 M Tris-HCl, pH 6.8. Store at 4°C.
4. 10% (w/v) SDS. Store at room temperature.
5. Ammonium persulfate (Bio-Rad): prepare a fresh 10% (w/v)
solution in water.
6. Running buffer (10×): 25 mM Tris, 192 mM glycine, 0.1%
(w/v) SDS. Store at room temperature.
7. Sample loading buffer (2×): 120 mM Tris-HCl pH 6.8, 20%
(v/v) glycerol, 4% (w/v) SDS, 40 mM dithiothreitol (DTT)
containing trace amount of bromophenol blue. To prepare this
solution, add 10 mL of 2 M DTT to 490 mL of the buffer that
had been prepared as a stock solution without the reducing
agent just before use. After use, store the remaining aliquot
at −20°C. The sample loading buffer without DTT can be
stored in 490 mL aliquots at room temperature.
8. Water-saturated butanol (50:50 v/v).
2.4. SDS
Polyacrylamide
Gel Electrophoresis
(SDS PAGE)
1. Rehydration solution: 7 M urea, 2 M thiourea, 4% (w/v)
CHAPS, with trace amount of bromophenol blue.
2. Immobilized pH gradient (IPG): 18-cm strips, 3-10 nonlinear
(GE Healthcare).
3. IPG buffer: 3-10 nonlinear (GE Healthcare).
2.5. First-Dimension
IEF
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