Biology Reference
In-Depth Information
6. Determine the protein concentration of the extract (
see
Note 12
).
7. Prepare aliquots of the wine protein extract and store them at
−80 °C.
The IPG strips are rehydrated and focused in an IPGphor
IsoElectric Focusing System (GE Healthcare).
3.3
IEF Parameters
1. For in gel active rehydration, load approximately 20
g of pro-
teins. Mix the corresponding volume of wine protein extract,
with 12.5
μ
L of glycerol and the remaining volume of rehydra-
tion solution for a fi nal volume of 125
μ
l (
see
Note 13
).
2. Pipet slowly the sample solution in the strip holder.
3. Place carefully the gel of the IPG strip in contact with the sam-
ple solution and proceed to the active rehydration of the IPG
strip with the IPGphor, at 30 V for 15 h.
4. At the end of the active rehydration step, place electrode paper
wicks between the IPG strips and the wire electrodes. Wet one
paper with ultrapure deionized water and insert the paper
between the IPG strip and the cathode. Wet another paper
with the rehydration solution and insert it between the IPG
strip and the anode (identifi ed by +). Cover the IPG strip with
mineral oil and initiate the focusing steps.
5. The following running conditions are applied according to the
pH gradient:
-pH 3-10, linear, 7 cm: 300 V for 15 min, 4,000 V linear
ramp for 2 h, 4,000 V for 10,000 Vh, 300 V as an addi-
tional step.
-pH 3-6, linear, 7 cm:, 50 V for 15 min, 250 V for 15 min,
4,000 V linear ramp for 2 h, 4,000 V for 10,000 Vh, 300 V
as an additional step.
6. At the end of IEF, remove the focused IPG strips from the
strip holder and place it, gel-side up, on a sheet of paper to
remove the excess of oil.
7. IPG strips can be used for the following equilibration steps or
stored at −80 °C until use.
μ
1. Place the IPG strip in a 10 mL screw-cap tube containing 5 mL
of reduction solution. Incubate 15 min at room temperature,
with gentle shaking.
2. Remove the IPG strip from the reduction solution and place it
in a tube containing 5 mL of alkylation solution. Incubate
15 min at room temperature with gentle shaking.
3. Blot the IPG strip, gel-side up, on a sheet of paper to remove
the excess of liquid and apply immediately the IPG strip on the
top of the SDS polyacrylamide gel (
see
Note 14
).
3.4 Equilibration
of Focused Proteins
