Biology Reference
In-Depth Information
1. Reswelling tray (GE Healthcare, Uppsala, Sweden).
2. Extraction solution for TCA/acetone precipitation (extraction
1): 20 % (w/v) trichloroacetic acid (TCA), 0.1 % (w/v) dithio-
threitol (DTT) in ice-cold acetone.
3. Rinsing solution for TCA/acetone precipitation (extraction
1): 0.1 % (w/v) DTT in ice-cold acetone.
4. SDS buffer (extraction 2): 4 % (w/v) Sodium dodecyl sulfate
(SDS), 5 % (w/v) sucrose, 0.3 % (w/v) DTT, 20 mM Na
2
HPO
4
,
pH 7.0.
5. Precipitation solution I (extraction 2): 10 mM DTT in ice-
cold acetone.
6. Polyvinylpolypyrrolidone (PVPP, Sigma, Bornem, Belgium).
7. Phenol (Tris-buffered, pH 8.0).
8. SDS-tris buffer (extraction 3): 2 % (w/v) SDS/30 % (w/v)
sucrose/0.1 M Tris-HCl/5 % (v/v)
2.1 Sample
Preparation
-mercaptoethanol, pH 8.0.
9. Precipitation solution II (extraction 3): Cold 0.1 M ammo-
nium acetate in methanol.
10. Rehydration buffer: 7 M urea, 2 M thiourea, 0.5/2 % (v/v)
pharmalyte or IPG buffer, 0.002 % (w/v) bromophenol blue,
2 % (w/v) 3-((3-cholamidopropyl)dimethylammonium)-
1-propanesulfonate (CHAPS).
11. pH test strips 4.5-10.0 pH, resolution: 0.5 pH unit (Sigma
Aldrich).
12. CyDyes (Cy2, Cy3, and Cy5) stock solution: Add 25
β
L dimeth-
ylformamide (DMF, less than 3 months old) to 25 nmol of dye.
μ
13. Labelling solution: 7 M urea; 2 M thiourea; 4 % (w/v) CHAPS;
30 mM Tris-HCL (
see
Note 1
).
14. Stopping solution: 10 mM lysine.
2.2 Isoelectro-
focusing
1. Sample buffer (2×): 7 M urea, 2 M thiourea, 4 % (w/v) CHAPS,
2 % (v/v) ampholyte, 2 % (w/v) DTT (
see
Notes 1
and
2
).
2. IPGphor (GE Healthcare, Uppsala, Sweden) or a similar elec-
trophoresis apparatus.
3. Manifold, paper wicks, sample cups.
4. Immobilized pH gradient strips: Gels strips may have different
lengths and pH gradients. In the present example, we present
the protocol for IPG strips of 24 cm on a pH gradient of 4-7.
5. Mineral oil (e.g., dry strip cover fl uid; GE Healthcare).
2.3 Second
Dimension—Vertical
System
1. Dalt twelve Gel Caster (GE Healthcare, Uppsala, Sweden).
2. Displacing solution: 0.375 M Tris-HCl, pH 8.8, 50 % (v/v)
glycerol, trace of bromophenol blue.
