Comparative Analyses of Protein Complexes by Blue Native DIGE - Difference Gel Electrophoresis (DIGE): Methods and Protocols - page 150

7. Combine the two labeled protein fractions in one Eppendorf

vessel.

8. Add 2 mL of blue loading buffer to the combined protein

fraction.

3.3. BN PAGE for First
Gel Dimension

1. Load the combined CyDye-labeled protein samples (see Sub-

heading 3.2 ) into a well of a BN gel (see Subheading 3.1 ).
2. Connect the gel unit to a power supply. Start electrophoresis at
a constant voltage (100 V for 45 min) and continue at a con-
stant current (15 mA for about 11 h). Electrophoresis should be
carried out at 4°C and in the dark in order to protect the dyes.

3. (Optionally): The BN cathode buffer, normally containing

Coomassie Blue, may be replaced after half of the electropho-
resis run against a BN cathode buffer without Coomassie Blue
(see Note 8).

BN PAGE can be combined with a second gel dimension, which is
carried out in the presence of SDS, to further separate the protein
complexes into their subunits. All published protocols for SDS
PAGE are suitable for combination with BN PAGE, e.g., the sys-
tem published by Laemmli ( 10 ). However, the Tricine-SDS PAGE
system developed by Schägger ( 11 ) generally gives the best resolu-
tion. The following instructions refer to this gel system carried out
in the Protean II gel unit (Bio-Rad). As mentioned above, gel elec-
trophoresis units of other manufacturers are of equal suitability:

3.4. SDS PAGE
for Second Gel
Dimension

1. Cut out a strip of a BN gel and incubate it in the denaturation

solution for 30 min at room temperature.

2. Wash the strip with ddH 2 O for 30-60 s. This step is important

because b-mercaptoethanol inhibits polymerization of
acrylamide.

3. Place the strip on a glass plate of an electrophoresis unit at the

position of the teeth of a normal gel comb.

4. Assemble the gel electrophoresis unit by adding 1-mm spacers,

the second glass plate and clamps, and transfer all into the gel
casting stand. The reduced thickness of the gel of the second
gel dimension (1 mm) in comparison to the strip of the fi rst gel
dimension (1.5 mm) avoids sliding down of the gel strip
between the glass plates in vertical position.

5. Prepare a 16% separation gel solution by mixing 12.4 mL of

acrylamide solution, 10 mL of SDS gel buffer, 7.6 mL ddH 2 O,
100 mL APS, and 10 mL TEMED, and pour the solution
into the space in-between the two glass plates below the BN
gel strip. Leave space for the sample gel solution for embed-
ding the BN gel strip. Overlay the separation gel with the
overlay solution. The gel should polymerize in about 60 min
(see Note 9).