Biology Reference
In-Depth Information
2.5 Components
for Protein Extraction:
Trichloroacetic Acid
Precipitation
1. Extracellular medium from
Vitis vinifera
SCC.
2. 2 % (w/v) polyvinylpolypyrrolidone (PVPP): Add 2 g PVPP to
100 mL of extracellular medium.
3. Trichloroacetic acid (TCA) solution: 24 % in water. Store at 4 °C.
1. Bio-Rad Protein Assay (dye reagent concentrate) (Bio-Rad)
(
see
Note 4
).
2. Protein standard: Bovine serum albumin (BSA) at a concen-
tration of 0.2 mg/mL in distilled water is used as a stock
solution. Store at −20 °C (
see
Note 5
).
2.6 Protein
Quantifi cation
2.7 Sodium Dodecyl
Sulfate-
Polyacrylamide Gel
Prepare the gel electrophoresis using a MiniProtean
®
3 Cell elec-
trophoresis kit (Bio-Rad).
1. Stacking gel buffer: 0.5 M Tris-HCl, pH 6.8. Store at 4 °C.
2. Resolving gel buffer: 1.5 M Tris-HCl, pH 8.8. Weigh 181.5 g
Tris. Add water to a volume of 1,000 mL. Mix and adjust pH
with 1 M HCl. Store at 4 °C.
3. 30 % Acrylamide/Bisacrylamide solution, 37.5:1 (Bio-Rad).
4. SDS solution: 10 % (w/v) in water. Store at room temperature.
5. Ammonium persulfate (APS) solution: 10 % (w/v) in water.
Store at room temperature (
see
Note 6
).
6.
N,N,N
-
Tetramethylethylenediamine (TEMED, Sigma).
Store at 4 °C (
see
Note 7
).
7. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE) running buffer: 25 mM Tris-HCl, pH 8.3,
192 mM glycine, 0.1 % SDS. Store at room temperature
(
see
Note 8
).
8. SDS-PAGE loading buffer: 0.5 M Tris-HCl, pH 6.8, 10 %
glycerol, 10 % SDS, 0.5 % bromophenol blue, 5 %
β
′
,N
′
-mercaptoethanol. Store at room temperature.
9. Prestained SDS-PAGE standards, broad range (Bio-Rad).
10. PowerPac 300 (Bio-Rad).
2.8 Two-Dimensional
Gel Electrophoresis
(IEF/SDS-PAGE)
1. Immobilized pH gradient (IPG) strips of pH range 3-10 and
13 cm length (GE Healthcare).
2. Rehydration solution with IPG buffer: 7 M Urea, 2 M thio-
urea, 4 % (w/v) CHAPS, IPG buffer pH 3-10 0.5 % (v/v),
50 mM dithiothreitol (DTT), and 0.002 % bromophenol blue
(
see
Note 9
). Weigh 0.84 g urea and add water to a volume of
1.2 mL. Dissolve and add 0.30 g thiourea, 0.08 g CHAPS,
0.016 g DTT, 10
L IPG buffer, and a few amount of bromo-
phenol blue and complete to 2 mL with water.
3. Reagents for SDS-PAGE are the same as described in
Subheading
2.7
(
items 2
-
7
).
μ
