Biology Reference
In-Depth Information
amount of proteins (<20 mg/L). On the other hand, 2D-E is still
a method of choice to have a detailed overview of the proteins
present in a wine and, is therefore better suited for a comparative
analysis than 1D-electrophoresis.
2
Materials
The following solutions are prepared from high purity chemicals
(i.e., electrophoresis grade) and ultrapure deionized water (e.g.,
18.2 M
). Disposable gloves are worn during each step to avoid
contaminations.
Ω
2.1
Wine Samples
A Chardonnay base wine elaborated by the Cooperative Nogent
l'Abbesse according to the traditional method was tested. The pro-
tein content was estimated around 10 mg/L (Bovine Serum
Albumin was used as a protein standard) (
see
Note 1
).
1. Standard rehydration solution (1×): 7 M Urea, 2 M Thiourea,
4 % (w/v) CHAPS, 0.5 % (v/v) IPG Buffer 3-10, traces of
Bromophenol blue, 60 mM DTT (
see
Note 2
).
2. Glycerol (Sigma Chemical Company, St. Louis, MO, USA).
Store at room temperature (
see
Note 3
).
3. Mineral oil (Sigma Chemical Company, St. Louis, MO, USA).
Store at room temperature.
4. Electrode wicks (Bio-Rad).
5. Readystrips™ IPG strips, pH 3-10 and pH 3-6, 7 cm
(Bio-Rad).
6. IPGphor Isoelectric Focusing System (Amersham Pharmacia
Biotech).
2.2 Isoelectro-
focusing Components
1. 1 M Tris-HCl, pH 8.8, buffer: Weigh 121.1 g of Tris and trans-
fer to a glass beaker. Add water to a volume of 900 mL. Mix and
adjust to pH 8.8 with HCl 6 N. Transfer the solution to a grad-
uated fl ask and make up to 1 L with water. Store at 4 °C.
2.3 SDS
Polyacrylamide Gel
Components
2. Acrylamide-Bis-acrylamide 37.5:1, 40 % (Bio-Rad). Store at
4 °C.
3. SDS 10 % (w/v) solution in water. Store at room
temperature.
4. Ammonium persulfate (APS) 10 % (w/v) solution in water
(
see
Note 4
).
5. TEMED, store at room temperature.
6. 1.2 M Bis-Tris/ 0.8 M HCl: Dissolve 12.55 g of Bis-Tris in
25 mL of water. Add 3.94 g of HCl 37 % (12 N) and adjust to
50 mL with water. Store at 4 °C.
