Comparative Analyses of Protein Complexes by Blue Native DIGE - Difference Gel Electrophoresis (DIGE): Methods and Protocols - page 149

4. Add TEMED and APS to the two gel solutions (95 mL 10%

APS/9.5 mL TEMED to the 4.5% gel solution and 61 mL
APS/6.1 mL TEMED to the 16% gel solution).

5. Pour the gradient gel, leaving space for the stacking gel, and

overlay with ddH 2 O. The gel should polymerize in about
60 min.

6. Pour off the ddH 2 O.

7. Prepare the stacking gel solution by mixing 1.5 mL of acrylam-

ide solution, 2.5 mL of BN gel buffer, and 11 mL ddH 2 O.

8. Add 65 mL APS and 6.5 mL TEMED and pour the stacking gel

around an inserted comb. The stacking gel should polymerize
within 30 min.

9. Prepare 1× BN anode buffer and 1× BN cathode buffer by

diluting the corresponding stock solutions.
10. Once the polymerization of the stacking gel is fi nished, care-
fully remove the comb.

11. Add the BN cathode and anode buffers to the upper and

lower chambers of the gel unit, respectively. Cool the unit
down to 4°C.

3.2. Sample
Preparation
for BN DIGE

Starting material can either be whole cells or isolated organelles.
Fractions should be treated under mild conditions in order to keep
proteins in their native conformation (avoid high salt, ionic deter-
gents, high temperatures, urea, etc.). All steps of the sample prepa-
ration should be carried out at 4°C. The BN gel should be prepared
before the sample preparation is started (see Subheading 3.1 ):

1. Cell or organelle fractions, including 100 mg protein each, are

centrifuged at full speed for 10 min at 4°C using an Eppendorf
centrifuge.

2. Sedimented material is resuspended in 10 mL of solubilization

buffer, pH 7.4, with digitonin and incubated for 20 min at
4°C (see Note 5).

3. Fractions are again centrifuged at full speed for 20 min at 4°C

using an Eppendorf centrifuge to remove insoluble material.

4. The supernatants containing solubilized protein complexes are

transferred into new 1.5-mL Eppendorf vessels and supple-
mented with 10 mL of solubilization buffer, pH 10, to adjust the
pH value of the protein solutions to about 8.5 (see Note 6).

5. Labeling reaction: For CyDye™ labeling reactions, 100 mg

protein of each sample is labeled with one CyDye by the addi-
tion of 1 mL of diluted CyDye solution to each protein fraction
(see Note 7). Centrifuge the samples briefl y and incubate for
30 min on ice in the dark.

6. Stop labeling reaction: Add 1 mL of lysine stock solution and

incubate samples for 10 min in the dark.