Comparative Analyses of Protein Complexes by Blue Native DIGE - Difference Gel Electrophoresis (DIGE): Methods and Protocols - page 148

6. SDS cathode buffer: 0.1 M Tris base, 0.1 M Tricine, 0.1%
(w/v) SDS, pH 8.25.
7. SDS anode buffer: 0.2 M Tris-HCl, pH 8.9.
8. Overlay solution: 1 M Tris-HCl, 0.1% (w/v) SDS, pH 8.45.
9. Denaturation solution: 1.0% (w/v) SDS, 1.0% (v/v)
b-mercaptoethanol.
10. SDS solution: 10% (w/v) SDS.
11. Protean II gel unit (Bio-Rad).

3. Methods

The following protocol is suitable for the comparison of protein
complexes of two related protein fractions. It is based on the usage
of the CyDye™ fl uor labeling reagents (GE Healthcare, Munich,
Germany). Both fractions should contain 100 mg protein and
should be labeled with different fl uorophores. Afterwards, the
samples are combined and loaded onto a single BN gel. The BN
gel should be prepared before sample separation.

Best resolution capacity of BN gels is achieved if the electropho-
retic separation distance is greater than 12 cm. The following
instructions refer to the Protean II electrophoresis unit (Bio-Rad,
Richmond, CA, USA; gel dimensions 0.15 × 16 × 20 cm). However,
units from other manufacturers are of comparable suitability for
BN PAGE, e.g., the Hoefer SE-400 or SE-600 gel systems (GE
Healthcare, Munich, Germany). Usage of gradient gels is recom-
mended because the molecular masses of protein complexes can
vary between 50 kDa and several 1,000 kDa (see Note 4):

3.1. Preparation
of a BN Gel

1. Prepare a 4.5% separation gel solution by mixing 2.4 mL of
acrylamide solution with 3.5 mL of BN gel buffer and 15.1 mL
ddH 2 O.
2. Prepare a 16% separation gel solution by mixing 7.4 mL of
acrylamide solution with 3 mL of BN gel buffer, 4.6 mL
ddH 2 O, and 3.5 mL glycerol.
3. Transfer the two gel solutions into the two chambers of a gra-
dient former and connect the gradient former via a hose and a
needle with the space in-between two glass plates which are
preassembled in a gel casting stand. Gradient gels can either be
poured from the top (16% gel solution has to enter the gel
sandwich fi rst) or from the bottom (4.5% gel solution has to
enter fi rst). For pouring the gel from the bottom, an adjustable
pump (e.g., Bio-Rad Econo Pump) is needed.