2D DIGE Analysis of Protein Extracts from Muscle Tissue - Difference Gel Electrophoresis (DIGE): Methods and Protocols - page 159

3. 10% ammonium persulfate: Prepare aliquots of 1 mL and store
at −20°C.
4. N , N , N , N ¢-tetramethylethylenediamine (TEMED). Store at 4°C.
5. SDS-PAGE running buffer (10×): 250 mM Tris, 1.92 M glycine,
and 1% SDS. For a fi nal volume of 1 L, weigh out 30.2 g of
Tris and 144.2 g of glycine and dissolve in about 600 mL
of water. Mix, fi lter the solution, add 10 g of SDS, and make
up to 1 L with water. Store at 4°C in a glass bottle.
6. 30% (v/v) isopropanol. Store at room temperature in a dark
glass bottle.
7. 0.5% agarose sealing solution: For a fi nal volume of 100 mL,
weigh out 0.5 g of agarose, add 100 mL of 1× SDS-PAGE
running buffer, and melt in a heating block or boiling water
(see Note 9).
8. Low-fl uorescence glass plates (GE Healthcare).
9. Gel casting cassettes.
10. Electrophoretic unit.

1. Typhoon variable mode imager (GE Healthcare) or similar.
2. DeCyder software (GE Healthcare) or similar.

2.4. Image Acquisition
and Analysis

3. Methods

3.1. Protein Extraction
from Muscle Tissue

1. At least 15-20 mg of muscle tissue is necessary to perform a
2D DIGE experiment.
2. Grind muscle tissue in a frozen mortar.
3. After sample disruption, add lysis buffer and sonicate the sam-
ple on ice (20-s bursts with 60-s rest between bursts) until no
particulate matter remains, or until no more of the particulate
matter is dissolved.
4. Centrifuge the sample at 12,000 × g for 5 min at 4°C to remove
any insoluble material.
5. Precipitate proteins in order to separate them from nonprotein
impurities (salts, nucleic acids, lipids, etc.).
6. Resuspend pellets with lysis buffer for DIGE.
7. Check the pH of the sample on ice. The sample pH must be
pH 8.0-9.0. Adjust the pH using dilute NaOH, if required.
8. Accurately quantify protein samples.

1. Remove the CyDye from the −20°C-freezer and leave to warm
for 5 min at room temperature.
2. After 5 min, add the specifi ed volume of DMF to each vial of
CyDye to prepare a CyDye stock solution (i.e., 5 mL of DMF

3.2. Protein Labeling