Biology Reference
In-Depth Information
3
6
Fig. 2
2D gel electrophoresis of champagne base wine proteins stained with
colloidal Coomassie Brilliant Blue. Wine proteins were separated by IEF in 7 cm
long pH 3-6 IPG strips, followed by SDS-PAGE in vertical 12 % gels
3.6 Gel image
Analysis
Images of the gels were acquired at 63.5
m resolution using the
GS-800 scanner and Quantity One v 4.62 software (Bio-Rad,
Hercules, CA). Spot detection and quantifi cation were performed
using the PDQuest Basic 8.0.1 software (Bio-Rad, Hercules, CA).
Reproducibility of 2D gels was assayed by running three replicates.
Selected protein features were modelled as Gaussians, and the rela-
tive optical densities (OD), i.e., the feature OD divided by the
total OD over the whole image, were computed. Means ± standard
deviation (
n
= 3) were calculated.
μ
4
Notes
1. A commercial wine or a wine elaborated in a laboratory can
also be used. Wines made with red grape varieties, and thus
containing an elevated content of phenolic compounds have
not been tested by this method. To eliminate these interfer-
ing compounds an additional step might be undergone by
adding PVPP.
2. Add DTT prior to use. This solution is stored in aliquots at
−80 °C.
3. Aliquots of glycerol (12.5
L correspond to 10 % (v/v) of the
fi nal rehydration volume. 125
μ
L is the fi nal rehydration vol-
ume needed for a 7 cm length strip) can be prepared and stored
at −20 °C.
4. Prepare the solution freshly.
5. 10 mL aliquots of the solution, without DTT or iodoacetamide,
are stored at −20 °C. Dissolution of DTT and iodoacetamide
can be diffi cult and may be achieved by a thorough shaking.
μ
