Biology Reference
In-Depth Information
7. Overlay agarose solution: 1 % (w/v) agarose low-melting,
0.2 % SDS, 0.15 M Bis-Tris/0.1 M HCl, 1
μ
g/
μ
L bromophe-
nol blue. Store at room temperature.
8. SDS Running buffer (1×): 25 mM Tris-HCl, pH 8.3, 192 mM
glycine, 0.1 % (w/v) SDS. The buffer is prepared from a 10×
stock commercial solution (Bio-Rad) and is refrigerated at
4 °C before use.
9. Mini-Protean 3 system, Spacer plates with 1.0 mm spacers
(Bio-Rad).
1. Reduction solution: 50 mM Tris-HCl, pH 8.8, 6 M urea,
30 % (v/v) glycerol, 2 % (w/v) SDS. Add 1 % (w/v) DTT prior
to use (
see
Note 5
).
2. Alkylation solution: 50 mM Tris-HCl, pH 8.8, 6 M urea, 30 %
(v/v) glycerol, 2 % (w/v) SDS, traces of bromophenol blue.
Add 2.5 % (w/v) iodoacetamide prior to use (
see
Note 5
).
2.4
Equilibration
2.5 Wine Protein
Extraction
1. Ultrafi ltration device with a 10 kDa membrane molecular
weight cut-off (MWCO) (
see
Note 6
).
2. Ethanol/TCA 15 % (w/v) solution: Prepare a fresh solution
before use by adding 15 g of trichloroacetic acid to 100 mL of
glacial ethanol (
see
Note 7
).
3
Methods
3.1 Wine
Ultrafi ltration
1. Using an ultrafi ltration device (
see
Note 8
), the wine sample is
concentrated ten times.
2. The ultrafi ltrate is discarded, and deionized water is added up
to the initial wine volume to dialyze the wine retentate.
3. Dialysis against deionized water is repeated one more time
(
step 2
).
4. The fi nal wine retentate is immediately precipitated or stored
at −80 °C.
1. Mix 5 mL of wine retentate with 40 mL of ethanol/TCA 15 %
(w/v) freshly prepared.
2. Store the sample at −20 °C during 1 h (
see
Note 9
).
3. Centrifuge 10 min at 9,500 ×
g
and 4 °C, then discard the
supernatant.
4. Wash the pellet with 20 mL of ethanol, to remove TCA
(
see
Note 10
) and centrifuge as described above. Repeat
this step again.
5. Dry the resulting pellet with nitrogen gas to remove ethanol
and solubilize the wine protein extract with 1 mL of standard
rehydration solution (
see
Note 11
).
3.2 Wine Protein
Precipitation
