In-gel digestion (Coomassie stained)
Notes:
1. Please always wear gloves and a lab coat to avoid keratin
contamination.
2. Please use non- autoclave tips or microcentrifuge tubes.
We strongly recommend using a brand new gel-casting system or a
disposable pre-cast gel for electrophoresis to eliminate contaminants
Reagents：
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Acetonitrile
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10 mM ammonium bicarbonate (NH4HCO3) / 50% acetonitrile buffer (pH 8.0)
20 mM ammonium bicarbonate
10 mM ammonium bicarbonate
Reduction solution: 10mM Dithiothreol (DTT, 1.54mg/ml) in 10 mM NH4HCO3
Alkylation solution: 55mM Iodoacetamide (IAM, 10.2mg/ml) in 10 mM
NH4HCO3
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50% acetonitrile/ 1% formic acid
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. Washing of gel pieces
a. Remove all liquid (such as water).
b. Wash the gel with 50 μl of 10 mM ammonium bicarbonate (NH4HCO3) / 50%
acetonitrile buffer (pH 8.0) for 15 min.(or vortex gentlely)
c. Remove buffer completely and add 100 μl acetonitrile to cover the gel
particles. The gels shrink and stick together.
d. Remove the acetonitrile and rehydrate the gel pieces with 25 μl 20 mM
ammonium bicarbonate for 5 min. (or vortex gentlely)
e. Add 25 μl acetonitrile and mix well.
f. Remove all liquid after 15 min of incubation. (or vortex gentlely)
※ If Coomassie blue is not removed, repeat step c to f.
g. Add 100 μl acetonitrile to cover the gel particles for 5 min. (or vortex
gentlely)
h. After the gel pieces have shrunk, 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 IAM/ 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.