Biology Reference
In-Depth Information
popular technique nowadays. Although BN PAGE is up to now
the technique which can be most easily applied to different sample
types (
11
), the use of Coomassie 250 G as charge carrier is limited.
As fl uorescence is quenched by the Coomassie dye, the combination
of BN PAGE with fl uorescence detection is impossible. Therefore,
BN PAGE is not compatible with CyDye detection. The following
protocol describes a modifi ed BN PAGE protocol which allows the
separation of plastid membrane protein complexes and detection
of chlorophyll and CyDyes by fl uorescence detection. Fluorescent
scanners used for visualization of fl uorescent dyes in DIGE, per-
fectly match for the detection of chlorophylls and their derivatives
as well. Hence, the classical DIGE technology can be extended in
plant research.
2. Materials
1.
Sample buffer: 10 mM Tris-HCl, pH 8.5, 10 mM MgCl
2
,
20 mM KCl. Store at 4°C.
2.1. Sample
Preparation
2.
CyDyes: dilute and store according to instruction manual.
3.
Lysine solution: 10 mM
L
-lysine. Store at 4°C.
4. Dodecyl maltoside solution: 10% (w/v) n-dodecyl-b-
D
-maltoside.
Store at −20°C.
5.
Digitonin solution: 10% (w/v) digitonin (see Note 1). Store
at −20°C.
6.
Lithium dodecyl sulfate buffer: 5% (w/v) lithium dodecyl
sulfate. Store at −20°C.
1.
Gel buffer (6×): 3 M e-amino caproic acid, 0.3 M Bis-Tris-HCl
pH 7.0. Store at 4°C (see Note 2). Adjust the pH of the Bis-Tris
solution at room temperature.
2.2. Casting of Native
Gradient PAGE Gels
2.
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.
3.
Glycerol (100%). Store at room temperature.
4. TEMED:
N
,
N
,
N
,
N
¢-tetramethyl-ethylenediamine. Store at room
temperature.
5. Ammonium persulfate (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.
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