Biology Reference
In-Depth Information
2. Materials
1. Cell lysis buffer: 30 mM Tris, 7 M urea, 2 M thiourea, 4%
(w/v) CHAPS, pH 8.0. Adjust using 1 M HCl and store in
aliquots at −20°C. The buffer is stable for at least 3 months.
2.1. Cell Lysis
1. Cy3 and Cy5 saturation dyes for cysteine labeling (GE
Healthcare Bio-Sciences, Uppsala, Sweden). Store at −70°C.
2. N , N -Dimethylformamide (DMF) anhydrous >99.8%. Do not
use longer than 3 months after opening.
3. Reconstituted solutions of Cy3 and Cy5 saturation dyes in
DMF at a 2 mM concentration.
4. 2 mM Tris-(2-carboxyethyl)phosphinehydrochloride (TCEP)
solution: dissolve 2.8 mg TCEP in 5 mL of water. Aqueous
TCEP solution is unstable and must be prepared fresh.
5. Sample buffer I: 7 M urea, 2 M thiourea, 4% (w/v) CHAPS.
Store in aliquots at −20°C. This buffer is stable for at least
6 months.
6. Sample buffer II: 7 M urea, 2 M thiourea, 4% (w/v) CHAPS,
130 mM dithiothreitol (DTT), 2% (v/v) Pharmalyte™ pH
3-10 (GE Healthcare Bio-Sciences, Uppsala, Sweden; Cat.
No. 17-0456-01). Prepare fresh by addition of Pharmalyte and
DTT to sample buffer I.
7. Protein Determination Reagent (e.g., USB, code 30098) for
determination of protein concentration.
8. Appropriate laser scanner (e.g., from the Typhoon series; GE
Healthcare Bio-Sciences, Uppsala, Sweden).
9. Appropriate software for image analysis and spot quantifi cation
(e.g., DeCyder 2D Differential Analysis software; GE
Healthcare Bio-Sciences, Uppsala, Sweden).
2.2. Reconstitution
of Dye and Protein
Labeling
3. Methods
Biological samples contain highly complex mixtures of completely
different biochemical macromolecules such as DNA/RNA, pro-
teins, and lipids. The distribution among these substance classes is
dependent on the biological source and may therefore require spe-
cial steps to avoid interference with 2D gel electrophoresis in gen-
eral or 2D DIGE saturation labeling, e.g., removal of excess amounts
of lipids or nucleic acids. In addition, some samples may contain
especially high amounts of hydrophobic proteins, the solubilization
3.1. Sample
Preparation for
Saturation DIGE
 
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