2. Add an appropriate volume (50 mL for mini-PROTEAN gels
8.6 cm  6.8 cm) of Oriole fluorescent gel staining solution
(BioRad) and follow the manufacturer instructions (see
Note 7).
3. Cover the container (Oriole is light sensitive) and leave it in a
shaker for 90 min. After staining, replace staining solution by
ultrapure water (gels can be preserved in water protected from
light at 4
C for several weeks before image analysis is
carried out).
4. Proteins stained with Oriole can be visualized (and also documented, if an appropriate video camera is incorporated) in any
imaging device that uses UV light excitation. Alternatively, the
safer option is the use of a blue light transilluminator (for
example, Blue-light DarkReader, Clare Chemical Research,
CO, USA).
5. Gel slides (fractions) are excised using a scalpel or razor blade
under a blue light transilluminator and deposited in a clean
microtube with HPLC-grade water (microtube I) (see Note 8).
3.5 In-Gel Protein
Digestion and Mass
Spectrometry Analysis
1. Dice the gel fractions (using a blade/scalpel) into several
1–2 mm
3 pieces and then wash twice in 100 μL of ultrapure
water for 10 min. Spin to move gel pieces down, removing out
all liquid after each washing cycle. At this point, the procedure
could be interrupted and gel plugs preserved for a long-term
(months) in 5% acetic acid solution at 4
C.
2. Add 100 μL (or a higher volume if necessary to completely
immerse the gel plugs) of HPLC-grade acetonitrile for 10 min
in order to dehydrate gel plugs. Use occasional vortexing.
Remove carefully acetonitrile solution without removing gel
plugs. Repeat again this step. Allow gel plugs to dry out in a
speed-vacuum.
3. Add 100 μL of fresh 10 mM dithiothreitol (DTT) solution and
incubate for 30 min at room temperature or 55
C (reaction
might work slightly better at this higher temperature according
to some reports) in order to reduce protein disulfide bonds.
Increase the DTT volume if necessary. Spin gel pieces down
and remove carefully the DTT solution. Check that reaction
pH is slightly alkaline; otherwise, DTT action will be less
effective (this also applies for IAM reaction; see below). If
necessary, prepare working DTT and IAM solutions in
25 mM ammonium bicarbonate.
4. Add 100 μL of fresh 100 mM iodoacetamide (IAM) solution
and incubate for 30 min at room temperature protected from
light in order to alkylate reduced cysteine residues and prevent
the formation of disulfide bonds. Increase the IAM volume if
necessary. Spin gel pieces down and remove carefully the IAM
solution.
86
Angel P. Diz and Paula Sa ´ nchez-Marı ´n
8.6 cm  6.8 cm) of Oriole fluorescent gel staining solution
(BioRad) and follow the manufacturer instructions (see
Note 7).
3. Cover the container (Oriole is light sensitive) and leave it in a
shaker for 90 min. After staining, replace staining solution by
ultrapure water (gels can be preserved in water protected from
light at 4
C for several weeks before image analysis is
carried out).
4. Proteins stained with Oriole can be visualized (and also documented, if an appropriate video camera is incorporated) in any
imaging device that uses UV light excitation. Alternatively, the
safer option is the use of a blue light transilluminator (for
example, Blue-light DarkReader, Clare Chemical Research,
CO, USA).
5. Gel slides (fractions) are excised using a scalpel or razor blade
under a blue light transilluminator and deposited in a clean
microtube with HPLC-grade water (microtube I) (see Note 8).
3.5 In-Gel Protein
Digestion and Mass
Spectrometry Analysis
1. Dice the gel fractions (using a blade/scalpel) into several
1–2 mm
3 pieces and then wash twice in 100 μL of ultrapure
water for 10 min. Spin to move gel pieces down, removing out
all liquid after each washing cycle. At this point, the procedure
could be interrupted and gel plugs preserved for a long-term
(months) in 5% acetic acid solution at 4
C.
2. Add 100 μL (or a higher volume if necessary to completely
immerse the gel plugs) of HPLC-grade acetonitrile for 10 min
in order to dehydrate gel plugs. Use occasional vortexing.
Remove carefully acetonitrile solution without removing gel
plugs. Repeat again this step. Allow gel plugs to dry out in a
speed-vacuum.
3. Add 100 μL of fresh 10 mM dithiothreitol (DTT) solution and
incubate for 30 min at room temperature or 55
C (reaction
might work slightly better at this higher temperature according
to some reports) in order to reduce protein disulfide bonds.
Increase the DTT volume if necessary. Spin gel pieces down
and remove carefully the DTT solution. Check that reaction
pH is slightly alkaline; otherwise, DTT action will be less
effective (this also applies for IAM reaction; see below). If
necessary, prepare working DTT and IAM solutions in
25 mM ammonium bicarbonate.
4. Add 100 μL of fresh 100 mM iodoacetamide (IAM) solution
and incubate for 30 min at room temperature protected from
light in order to alkylate reduced cysteine residues and prevent
the formation of disulfide bonds. Increase the IAM volume if
necessary. Spin gel pieces down and remove carefully the IAM
solution.
86
Angel P. Diz and Paula Sa ´ nchez-Marı ´n