Biology Reference
In-Depth Information
2. Materials
Buffers, solutions, and reagents are given in the order of usage
according to the methods protocol. Prepare all solutions freshly
using analytical grade chemicals in combination with pure deion-
ized water.
2.1. Preparation
of a BN Gel
1.
Acrylamide solution: 40%, acryl/bisacryl = 32/1 (AppliChem,
Darmstadt, Germany).
2.
BN gel buffer (6×): 1.5 M aminocaproic acid, 150 mM BisTris,
pH 7.0 (adjust at 4°C).
3.
N
,
N
,
N
¢,
N
¢-tetramethylethylenediamine (TEMED), 99% (Sigma,
St. Louis, Missouri, USA).
4.
Ammonium persulfate solution (APS): 10% (w/v) ammonium
persulfate.
5.
BN cathode buffer (5×): 250 mM tricine, 75 mM BisTris, 0.1%
(w/v) Coomassie G 250, pH 7.0 (adjust at 4°C).
6.
BN anode buffer (6×): 300 mM BisTris, pH 7.0 (adjust
at 4°C).
7.
Protean II gel unit (Bio-Rad, Richmond, Ca, USA).
2.2. Sample
Preparation
for BN DIGE
1.
Solubilization buffer, pH 7.4, with digitonin: 30 mM HEPES,
150 mM potassium acetate, 10% (v/v) glycerol, pH 7.4, sup-
plemented with 5% digitonin (see Note 1). Solution should be
briefl y boiled for dissolving digitonin. Buffer is stored at 4°C.
2.
Solubilization buffer, pH 10: 30 mM HEPES, 150 mM potas-
sium acetate, 10% (v/v) glycerol, pH 10 (see Note 2).
3. CyDye™ fl uor minimal labeling reagents: Cy2™, Cy3™, and
Cy5™ (GE Healthcare, Munich, Germany). The fl uorophores
(400 pmol) are diluted in dimethylformamide (DMF) according
to the manufacturers' instructions. Diluted CyDyes are stored at
−20°C and should be used within 3 months (see Note 3).
4.
Lysine stock solution: 10 mM lysine, stored at 4°C.
5.
Blue loading buffer: 750 mM aminocaproic acid, 5% (w/v)
Coomassie 250 G, stored at 4°C.
1. Acrylamide solution: 40%, acryl/bisacryl = 32/1 (AppliChem).
2.
N
,
N
,
N
¢,
N
¢-tetramethylethylenediamine (TEMED), 99%
(Sigma).
2.3. SDS PAGE
for Second Gel
Dimension
3.
APS: 10% (w/v) ammonium persulfate.
4.
SDS gel buffer: 3 M Tris-HCl, 0.3% (w/v) SDS, pH 8.45.
5.
BN gel buffer BN (6×): see Subheading
2.1
.
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