Comparative Analyses of Protein Complexes by Blue Native DIGE - Difference Gel Electrophoresis (DIGE): Methods and Protocols - page 151

6. Prepare a 10% sample gel solution by mixing 2.5 mL of

acrylamide solution, 3.4 mL of BN gel buffer, 1 mL glycerol,
100 mL of SDS solution, 2.9 mL ddH 2 O, 83 mL APS, and
8.3 mL TEMED.

7. Discard the overlay solution and pour the sample gel embed-

ding the BN gel lane. The casting stand should be held slightly
diagonally to avoid air bubbles to be captured underneath the
BN gel lane (see Note 10).

8. Add the SDS cathode and anode buffers to the upper and

lower chambers of the gel unit.

9. Connect the gel unit to a power supply. Carry out electropho-

resis at 30 mA/gel over night. The gel run should take place in
the dark to protect the fl uorophores.

After completion of gel electrophoresis, fl uorophores can be
detected in 1D BN or 2D BN/SDS gels using a fl uorescence scan-
ner (e.g., the Typhoon fl uorescence scanner from GE Healthcare)
(see Fig. 1 ). Keep gels in the dark before starting the scanning

3.5. Visualization
of CyDye™-Labeled
Protein

Fig. 1. Comparative analysis of the mitochondrial proteomes of a wild-type (Col-0) and a
mutant (Col-0- D At1g47260) cell line of the model plant Arabidopsis thaliana . Proteins of
the Col-0 fraction were labeled with Cy3 and proteins of the Col-0- D At1g47260 fraction
with Cy5. Combined protein fractions were separated by 2D BN/SDS PAGE, and protein
visualization was carried out by laser scanning at the respective wavelengths using the
Typhoon laser scanner. On the resulting overlay image, Cy3-labeled proteins are indicated
in green and Cy5-labeled proteins in red . Proteins of equal abundance in the two com-
pared fractions are yellow . The molecular masses of standard proteins are given to the
right of the fi gure; the identities of protein complexes are given below the gel. I complex I;
V complex V; III 2 dimeric complex III; I + III 2 supercomplex containing complex I and dimeric
complex III. The mutant line lacks a subunit of complex I. As a consequence, amounts of
complex I and the I + III 2 supercomplex are drastically reduced (from Perales et al. ( 3 ) ,
modifi ed).