Biology Reference
In-Depth Information
6. Use a centrifugal fi lter device to ultrafi ltrate 15 mL of wine or,
if feasible, higher volume ultrafi ltration systems, in order to
obtain a suffi cient volume of retentate.
7. Ethanol can be stored at −20 °C. TCA must be added prior use
since the solution is unstable at −20 °C and might become yel-
low after storage. TCA is extremely hazardous, thus it is impor-
tant to avoid inhalation, skin and eyes contacts. Furthermore,
TCA is highly hygroscopic, thus it is important to store the
product in an appropriate place in order to weigh the correct
amount of TCA.
8. A hydrophilic polysulfone membrane (30 UFIB, Setric Genie
Industriel, SGI, France) with a 10 kDa molecular weight cut-
off was used. Wine ultrafi ltration was carried out at 12 °C
using a crossfl ow fi ltration module coupled with the SGI
Hi-Flow system (pumping system plus glass tank). Alternative
ultrafi ltration device (with an equivalent molecular weight cut-
off) might be used, as for example centrifugal fi lter devices.
9. Alternatively, the solution can be left overnight at −20 °C. In
this case, TCA might be more diffi cult to eliminate and an
additional washing step might be needed ( step 4 ,
Subheading 3.2 ). The mentioned volumes can be modifi ed,
even if it is better to keep these proportions: one volume of
wine concentrate diluted with eight volumes of ethanol/TCA.
10. The volume of ethanol must be at least 20 mL, to maximize
the removal of TCA. Precipitation with TCA has both advan-
tage and disadvantage. It allows proteases inactivation, reduces
salt concentration but interferes with IEF leading to horizontal
streaking.
11. In this experiment, the protein pellet was mixed with 1 mL of
rehydration buffer. A lower volume, as for example 500
L,
might be used if all the proteins are well solubilized. It also
depends on the initial protein concentration of the wine
sample.
12. The protein content was estimated with the Bradford method
as modifi ed by Fey et al. [ 30 ], using Bovine Serum Albumin as
protein standard.
13. The protein load is adapted according to the length of the strip
and the staining method used.
14. The second dimension gel can be prepared 24 h in advance
and stored in the casting gel cassette with a layer of deionized
water to exclude the drying of the gel.
15. Use forceps and a spatula to place correctly the IPG strip.
Avoid trapping air bubbles between the IPG strip and the sec-
ond dimension gel. The gel-side of the IPG strip must be in
contact with the little glass plate.
μ
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