In-gel digestion (Silver stained)
Notes:
1. Please always wear gloves and lab coat to avoid keratin
contamination
2. Please use cleaned tips or microcentrifuge tubes but do not
autoclave
We strongly recommend using a brand new gel-casting system or a
disposable pre-casted gel for electrophoresis to eliminate contaminants
This protocol was referred to Electrophoresis 1999, 20, 601-605
Reagents：
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Acetonitrile
10 mM ammonium bicarbonate
30 mM potassium ferricyanide, K3[Fe(CN)6]
100 mM sodium thiosulfate, Na2S2O3·5H2O
Reduction solution: 10mM Dithiothreol (DTT, 1.54mg/ml) in 10 mM NH4HCO3
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Alkylation solution: 55mM Iodoacetamide (IAA, 10.2mg/ml) in 10 mM
NH4HCO3
10 mM ammonium bicarbonate (NH4HCO3) / 50% acetonitrile buffer (pH 8.0)
50% acetonitrile/ 1% formic acid
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Procedure：
A. Excision of protein bands from polyacrylamide gels
a. Excise the spots of interest from the gel (1 mm3 pieces).
b. Transfer the gel cube to a cleaned but not autoclaved 0.5 ml microcentrifuge
tube.
B. Destaining the gel pieces
a. Remove all liquid from the gel pieces.
b. Prepare the destain working solution by mixing a 1:1 ratio of 30 mM
potassium ferricyanide and 100 mM sodium thiosulfate. (working solution is
unstable, please prepare freshly)
c. Add 30-50μl of working solution to cover the gel, incubate at RT with vortex
till the brownish color disappeared.
d. Discard the working solution and wash the gel 3-5 times with 50 μl ddH2O to
stop the reaction.
e. Add 20 mM NH4HCO3 and incubate at RT for 20 min to remove the yellowish
color.
f. Repeat step e. until the gel pieces become colorless.
g. Remove all remaining liquid and add 100 μl acetonitrile to cover the gel and
incubate for 5 minutes. Repeat the step again until the gel pieces become
opaque white.
h. Remove the acetonitrile.
i. Dry down gel in a vacuum centrifuge or air dry.
C. Reduction and alkylation
a. Add 50 μl reduction solution (10 mM DTT/ 10mM ammonium bicarbonate)
(freshly prepared).
b. Incubate for 15 min at 56°C, then chill tubes to room temperature.
c. Remove DTT completely and add 50 μl freshly prepared alkylation solution
(55 mM IAA/ 10 mM ammonium bicarbonate). * Light sensitive!!
d. Incubate for 20 min at room temperature in the dark.
e. Remove alkylation solution.
f. Wash the gel with 50 μl 10 mM ammonium bicarbonate / 50% acetonitrile
buffer (pH 8.0) for 15 min, 1 or 2 times.
g. Add 100 μl acetonitrile to cover the gel particles.
h. After the gel pieces have shrunk, remove the acetonitrile.
i. Dry down the gel particles in a vacuum centrifuge or air dry.
D. In-gel digestion
a. Add 20-30 μl freshly prepared enzyme solution (10 ng/μl of trypsin, in cold
10mM ammonium bicarbonate) to cover the gel.
※The trypsin powder should be reconstituted in 50mM acetic acid to 1-2
μg/μl and stored at -20℃ as described in manufacturer’s manual.
b. Incubate at 4°C for 60 min.
c. Add 20-30 μl 10 mM NH4HCO3 to keep the gel wet overnight, but avoid
excess liquid. (Make sure that the lid is tightly closed).
d. Incubate at 37°C overnight.
E. Extraction of peptides
a. Harvest trypsin digests into a new tube.
b. Add 30-50 μl of 50% acetonitrile/ 1% formic acid to each gel.
c. The tube is sonicated for 10 min.(Optional)
d. Pool the supernatant containing peptide mixtures to the same tube (step a).
e. Repeat the step b-d.
f. Concentrate in a Speed Vac.