Bio-Rad GS-900™ Calibrated Densitometer - Manual (Page 56)

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2-D Electrophoresis Guide Methods Chapter 12: In-Gel Trypsin Digestion 2-D Electrophoresis Guide
Mix Solutions A and B in a 1:1 ratio.
This is the silver destain solution.
Place each gel plug in a 0.5 ml or
1.5 ml plastic tube.
Add 50 µl of silver destain solution.
Incubate 20 min at room temperature.
Using a laboratory pipet, remove the
silver destain solution and add 50 µl
of fresh solution.
Repeat steps 2 and 3 for a total
of three treatments. Following the
last incubation, remove the silver
destain solution.
Proceed with the procedure
described below.
Add 100 μl of destaining buffer to
the gel plug and incubate for 30 min.
Remove and discard the solution.
Repeat step 1 two more times.
Add 400 μl of destaining buffer to
the gel plug and incubate overnight
at room temperature.
Remove destaining buffer and
dehydrate the gel by adding 50 μl
of acetonitrile. Incubate 10 min
at room temperature and remove
excess solution (for example,
by aspiration).
Dry the gel piece for 30 min in a
laminar flow hood.
Add 100 μl of reducing solution to the
gel plug and incubate 30 min at room
temperature. Remove excess liquid.
Add 100 μl of alkylating solution to
the gel plug and incubate 30 min
at room temperature in the dark.
Remove excess liquid.
Add 50 μl of acetonitrile to the gel
plug and incubate for 10 min at room
temperature. Remove excess liquid
and proceed to digestion.
To the dried gel plug, add a volume
of trypsin solution equivalent to the
volume of the original hydrated plug
(1.5 mm plug = 3.4 μl).
Incubate at room temperature for
10 min (center of gel will change from
opaque to clear). If gel plugs aren’t
swollen, add a few more μl of trypsin
solution and incubate for an additional
10 min.
Add enough 50 mM NH
4
HCO
3
to cover the gel plug (~10 μl).
Incubate at 37°C for at least 3 hr.
Remove trypsin solution from the
gel plug, and store it in another vial.
To the gel plug, add 2–8 µl of extraction
solvent. For MALDI-MS analysis,
keep this volume as small as possible
(2–3 µl). For LC-MS analysis, add 8 µl.
Incubate 30 min at room temperature.
Combine extraction solvent with
trypsin solution.
General Destaining Protocol
Reduction and Alkylation Protocol
This step is not necessary for 2-D gel plugs if they
have already been reduced and alkylated during the
sample preparation or equilibration steps.
Digestion Protocol
Extraction Protocol
1
1
1
1
1
2
2
2
2
2
3
3
3
4
4
4
5
6
5
3
3
4
This protocol for tryptic digestion of gel pieces (plugs)
excised from SDS-PAGE gels is derived from the
protocol described by Speicher et al. (2000). It can
be used in conjunction with any of the non-silver
stains described in this guide.
Reagents and Solutions
■ ■
Ammonium bicarbonate, NH
4
HCO
3
■ ■
Acetonitrile
■ ■
Iodoacetamide
■ ■
HPLC-grade water (for example, Burdick and
Jackson AH365)
■ ■
Trifluoracetic acid (TFA) (for example,
Thermo Scientific 28904)
■ ■
Octyl b-D-glucopyranoside (for example,
Sigma Aldrich 08001)
■ ■
Sequencing-grade modified trypsin, porcine
(for example, Promega V5111)
Destaining buffer (50:50 ACN:0.2 M NH
4
HCO
3
)
Dissolve 158 mg of NH
4
HCO
3
in 5 ml HPLC-grade
water and add 5 ml acetonitrile.
50 mM NH
4
HCO
3
Dissolve 79 mg of NH
4
HCO
3
in 20 ml of water.
Reducing solution
Dissolve 555 mg of DTT in 3 ml of 50 mM NH
4
HCO
3
.
Alkylating solution
Dissolve 54 mg of iodoacetamide in 3 ml of 50 mM
NH
4
HCO
3
.
Trypsin solution (20 μg/ml)
Dissolve 20 μg of trypsin in 1 ml of 50 mM NH
4
HCO
3
.
Extraction solvent
Combine 950 µl of 1% TFA with 50 µl of 1% octyl
D-glucopyranoside.
Destaining Gel Plugs from Silver-Stained
Gels (Pre-Treatment)
Gels that have been stained with a mass
spectrometry–compatible silver stain benefit
from an additional treatment to remove silver
metal by oxidation.
All materials used should be ACS reagent grade
or better.
Solution A (30 mM potassium ferricyanide)
To prepare 50 ml, dissolve 494 mg of potassium
ferricyanide [K
3
Fe(CN)
6
] in 50 ml of water. This solution
may be stored indefinitely at room temperature.
Solution B (100 mM sodium thiosulfate)
To prepare 50 ml, dissolve 791 mg of anhydrous sodium
thiosulfate [Na
2
S
2
O
3
] in 50 ml of water. This solution may
be stored for one year in a sealed bottle.
Protocol
Prepare the silver destain solution just prior to use.
It is good for only one use. Discard any excess.
Tryptic Digestion Protocol