Biology Reference
In-Depth Information
2. Materials
The use of high-quality electrophoresis/proteomic-grade chemicals
is paramount to achieving successful experiments with resulting
identifi cations—this is especially true of the quality of the water
used in all buffers and solutions and should be of 18 M
Ω
or less.
2.1. First Dimension
of 2D Electrophoresis
1. Rehydration buffer: 7 M urea, 2 M thiourea, 4% (w/v) CHAPS,
40 mM dithiothreitol (DTT), 0.5% (v/v) carrier ampholyte.
2. Immobilized pH gradient (IPG) strips (see Note 1).
1. SDS equilibration buffer: 6 M urea, 30% glycerol, 2% sodium
dodecyl sulfate (SDS), 0.002% bromophenol blue, 75 mM Tris-
HCl, pH 8.8 (step 1: DTT followed by step 2: iodoacetamide).
2. SDS gel: acrylamide (10%), bisacrylamide (3%), SDS (0.1%),
ammonium persulfate, TEMED, 0.37 M Tris-HCl, pH 8.8
(see Note 1).
3. SDS running buffer: 25 mM Tris-base, 192 mM glycine, 0.2%
SDS.
4. Bind-Silane (see Note 2).
2.2. Second Dimension
of 2D Electrophoresis
2.3. Labeling (Minimal
Dye Approach)
1. CyDye DIGE fl uors (Cy2, Cy3, and Cy5).
2. Dimethyl formamide (DMF) (see Note 3).
3. Labeling buffer: 7 M urea, 2 M thiourea, 4% CHAPS, 30 mM
Tris-HCl, pH 8.5.
4. 10 mM lysine.
5. pH test paper 7.5-9.5 (see Note 4).
6. 50 mM NaOH.
1. Ethanol.
2. Glacial acetic acid.
3. Deionized water.
4. Fluorescent stain (e.g., Deep Purple™, SYPRO ® Ruby).
2.4. General Reagents
1. First dimension electrophoresis unit.
2. Second dimension electrophoresis unit.
3. Power supply.
4. Temperature controlled recirculating water bath.
5. Ice bucket/ice.
6. Imaging device (laser scanner).
7. Analysis software.
2.5. General Apparatus
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