13. Remove all leftover 1:1 ethanol/ethyl acetate by drying 2 min
in a vacuum dryer (see Note 43).
14. Add 125 μL of 2D buffer containing DTT to the final pellet
and vortex. Complete the resuspension of protein pellet by
agitation at maximum rpm or sonication (see Note 44).
15. Measure protein concentration as instructed by Bradford and
continue with the IEF. Stored at À80
C until use.
3.3 First Dimension:
Isoelectrofocusing
(IEF)
1. Choose the proper IPG strip length and pH range (see
Note 45). Prepare rehydration solution in the appropriate
volume indicated in the manufacturer’s instructions for each
IPG strip: take the volume of FTSC-labeled sample equivalent
to the maximum amount of protein that can be loaded onto the
IPGs and add 2D buffer up to the appropriate rehydration
solution volume.
2. Pipette the appropriate volume of rehydration solution into
each strip holder and distribute homogeneously between the
two electrodes. Remove any air bubbles.
3. Extract gently IPG strips from the packed using flatted forceps
and remove the protective cover foil (see Note 46). Position the
IPG strip gel, with the gel side down, onto the strip holders
with the rehydration solution. Let it soak for at least 20 min (see
Note 47). Then, add mineral oil (see Note 48) onto the top of
the strip, cover it with the cap and allow the rehydration of the
gel in the dark for the time indicated in instructions (10–20 h).
4. Perform protein focusing by applying the voltage/time profiles
suggested by the manufacturer.
5. Strips can be directly used or frozen at À80
C instead (see
Note 49).
3.4 Second
Dimension: SDS
Polyacrylamide Gel
1. Prepare six 26 Â 20 cm running gels (10% T, 2.6% C) (see Note
50) by mixing 137.2 mL of resolving gel buffer, pH 8.8,
137.2 mL 40% acrylamide, 73.6 mL 2% bisacrylamide,
5.5 mL of 10% SDS, and 196.5 mL of deionized H 2 O. Add
2 mL of 10% APS and 200 μL of TEMED, then mix again (see
Note 51). Sonicate the buffers for 15 min to degas the solution
in an ultrasonic bath (see Note 52).
2. Quickly fill the empty gel caster with running gel mixture up to
2–3 cm to the end of the glasses (see Note 53).
3. Gently add about 20 mL isopropanol and let set at least 1 h.
4. Rinse isopropanol out with H 2 O and store gels overnight at
4
C to achieve complete gel polymerization (see Note 54).
5. Carefully remove the IPG gels from the strip holder using
forceps and introduce them in a Falcon-like tube containing
the reducing solution (5–15 mL) (see Note 55).
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Lucı ´a Me ´ ndez et al.
in a vacuum dryer (see Note 43).
14. Add 125 μL of 2D buffer containing DTT to the final pellet
and vortex. Complete the resuspension of protein pellet by
agitation at maximum rpm or sonication (see Note 44).
15. Measure protein concentration as instructed by Bradford and
continue with the IEF. Stored at À80
C until use.
3.3 First Dimension:
Isoelectrofocusing
(IEF)
1. Choose the proper IPG strip length and pH range (see
Note 45). Prepare rehydration solution in the appropriate
volume indicated in the manufacturer’s instructions for each
IPG strip: take the volume of FTSC-labeled sample equivalent
to the maximum amount of protein that can be loaded onto the
IPGs and add 2D buffer up to the appropriate rehydration
solution volume.
2. Pipette the appropriate volume of rehydration solution into
each strip holder and distribute homogeneously between the
two electrodes. Remove any air bubbles.
3. Extract gently IPG strips from the packed using flatted forceps
and remove the protective cover foil (see Note 46). Position the
IPG strip gel, with the gel side down, onto the strip holders
with the rehydration solution. Let it soak for at least 20 min (see
Note 47). Then, add mineral oil (see Note 48) onto the top of
the strip, cover it with the cap and allow the rehydration of the
gel in the dark for the time indicated in instructions (10–20 h).
4. Perform protein focusing by applying the voltage/time profiles
suggested by the manufacturer.
5. Strips can be directly used or frozen at À80
C instead (see
Note 49).
3.4 Second
Dimension: SDS
Polyacrylamide Gel
1. Prepare six 26 Â 20 cm running gels (10% T, 2.6% C) (see Note
50) by mixing 137.2 mL of resolving gel buffer, pH 8.8,
137.2 mL 40% acrylamide, 73.6 mL 2% bisacrylamide,
5.5 mL of 10% SDS, and 196.5 mL of deionized H 2 O. Add
2 mL of 10% APS and 200 μL of TEMED, then mix again (see
Note 51). Sonicate the buffers for 15 min to degas the solution
in an ultrasonic bath (see Note 52).
2. Quickly fill the empty gel caster with running gel mixture up to
2–3 cm to the end of the glasses (see Note 53).
3. Gently add about 20 mL isopropanol and let set at least 1 h.
4. Rinse isopropanol out with H 2 O and store gels overnight at
4
C to achieve complete gel polymerization (see Note 54).
5. Carefully remove the IPG gels from the strip holder using
forceps and introduce them in a Falcon-like tube containing
the reducing solution (5–15 mL) (see Note 55).
236
Lucı ´a Me ´ ndez et al.