2D DIGE Analysis of Protein Extracts from Muscle Tissue - Difference Gel Electrophoresis (DIGE): Methods and Protocols - page 157

CyDye fl uors are then visualized separately using an imager
containing appropriate laser wavelengths. Digital images of each
sample will be analyzed by dedicated software. One of the most
important advantages of 2D DIGE is the introduction of the inter-
nal standard ( 8, 9 ) created by pooling an aliquot of all biological
samples and labeled with one of the CyDye DIGE fl uors (usually
Cy2 in a three-color experiment or Cy3 in a two-color experi-
ment). This means that every protein from all samples will be rep-
resented in the internal standard. Linking every sample in-gel to a
common internal standard gives some advantages compared to
conventional 2DE; in particular, it allows an accurate quantifi ca-
tion and the separation of experimental from biological variation,
and statistical analysis of spots between gels can be applied ( 10 ).

2. Materials

All solutions have to be prepared using ultrapure water (18 MWcm
at 20°C) and analytical grade reagents. Filtering all solution is
strongly recommended.

1. Homogenizer for sample disruption (see Note 1). 2.1. Sample

Preparation
and Quantitation
(Compatible
with CyDye DIGE
Minimal Labeling)

2. Lysis buffer: 30 mM Tris, 7 M urea, 2 M thiourea, 4% (w/v)

CHAPS, pH 8.5. For a fi nal volume of 100 mL, dissolve
364 mg of Tris, 42 g of urea, 15.2 g of thiourea, and 4 g of
CHAPS in about 50 mL of water. Adjust the pH with dilute
HCl and make up to 100 mL with water. Prepare aliquots of
1 mL and store at −20°C. Prior to use, add 1 mL of PMSF
10 mM (see Note 2).

3. Sonicator (see Note 3).

4. Precipitation method to purify protein sample to remove non-

protein impurities (see Note 4).

5. pH indicator strips (pH 4.5-10.0) to check the pH of the pro-

tein extract and sodium hydroxide (50 mM) to adjust the pH.

6. Quantitation method to determine sample concentration

(see Note 5).

2.2. Protein Labeling

1. CyDye™ DIGE fl uor Cy2, Cy3, and Cy5 minimal dye (GE

Healthcare) (see Note 6).

2. 99.8% DMF (anhydrous dimethylformamide) for the reconsti-

tution of CyDye, less than 3 months old from the day of
opening (see Note 7).

3. 10 mM lysine solution to stop the labeling reaction. The solu-

tion can be stored at 4°C for several months.