Native DIGE of Fluorescent Plant Protein Complexes - Difference Gel Electrophoresis (DIGE): Methods and Protocols - page 345

2.3. Casting of SDS
PAGE Gels

1. Separating gel buffer (8×): 3 M Tris-HCl (pH 8.8) Store at 4°C
(see Note 2).
2. Stacking gel buffer (2×): 0.25 M Tris-HCl (pH 6.8). Store at 4°C
(see Note 2). Add 60 mL of a 2% (w/v) bromphenol blue
solution to 100 mL of stacking gel buffer.
3. Acrylamide solution: 30% (w/v) acrylamide/bisacrylamide
solution (37.5:1, 2.6% C), acts in unpolymerized state as a
neurotoxin. Store as stated by the manufacturer.
4. TEMED. Store at room temperature.
5. APS: 10% (w/v) solution. Stable at 4°C for up to 2 weeks.
6. Water-saturated isobutanol: 50% (v/v) isobutanol. Store at
room temperature.

2.4. Transfer of Native
Gel Stripes to SDS
PAGE Gels

1. Solubilization buffer: 2% (w/v) sodium dodecyl sulfate, 66 mM
Na 2 CO 3 , 2% (v/v) b-mercaptoethanol. Store at 4°C for up to
1 month or prepare freshly (see Note 3).
2. Overlay solution: 0.5% (w/v) low melting agarose in 1× SDS
running buffer. Store at room temperature.

2.5. Electrophoresis

1. Native PAGE (fi rst dimension)
(a) Running buffer cathode (5×): 400 mM tricine, 75 mM
Bis-Tris-HCl (pH 7.0), 0.01% (w/v) lithium dodecyl
sulfate. Store at −20°C for long time storage or at 4°C for
some days. Adjust pH of buffer at room temperature.
(b) Running buffer anode (10×): 500 mM Bis-Tris-HCl
pH 7.0. Store at 4°C.
2. SDS PAGE (second dimension) running buffer cathode/
anode (10×): 250 mM Tris, 1.92 M glycine, 1% (w/v) sodium
dodecyl sulfate. Store at room temperature.

3. Methods

The protocol described below combines CyDye with chlorophyll
fl uorescence detection (see Note 4). In general, native PAGE (fi rst
dimension) is intended to separate protein complexes in a native
state. Therefore, it is advisable to carry out all steps of sample prep-
aration on ice to avoid protein complex degradation. This native
PAGE system separates protein complexes depending on the
volume/size of the single protein complexes. Therefore, the acryl-
amide concentration of the gel is responsible for the separation
range. Instructions stated below correspond to a 6-12% linear
gradient separating gel and a 4% stacking gel for the fi rst native
dimension which allows the separation of protein complexes in the