An Overview of 2D DIGE Analysis of Marine (Environmental) Bacteria - Difference Gel Electrophoresis (DIGE): Methods and Protocols - page 361

FLUORESCENCE LABELING

3 x R1 3 x R2 3 x R3 3 x R4

Reference
state
(R)

Unlabeled
extracts

3 x R1 3 x R2 3 x R3 3 x R4

Internal
standard
(IS)

Cy2-labeled
extract mixture

IS = R(1-4) + A(1-4) + B(1-4) + C(1-4)

Unlabeled
extracts

A2 A3 A4

A1 B1 B2 B3 B4 C1 C2 C3 C4

Test
states
(A, B, C)

A1 A2 A3 A4 B1 B2 B3 B4 C1 C2 C3 C4

PREPARATION OF MIXTURES

R1 R2 R3 R4 R1 R2 R3 R4 R1 R2 R3 R4

+ + + + + + + + + + + +

IS IS IS IS IS IS IS IS IS IS IS IS

+ + + + + + + + + + + +

A1 A2 A3 A4 B1 B2 B3 B4 C1 C2 C3 C4

2DE

B4 C4

A4

B3 C3

A3

B2 C2

A2

B1 C1

A1

IMAGE ACQUISITION (3-mode scanning)

Green (532 nm) Blue (488 nm) Red (633 nm)

Cy3-image Cy2-image Cy5-image

IMAGE ANALYSIS (DeCyder)

Raw data
12 gels
= 36 gel images

DIA
intragel comparison
1 gel

BVA
intergel comparison
12 gels

Fig. 3. Scheme of a typical 2D DIGE experiment consisting of fl uorescence labeling, preparation of mixtures of labeled
protein extracts, 2DE-based protein separation, image acquisition, and analysis (modifi ed from ( 31 ) ) .