Analysis of Proteins Using DIGE and MALDI Mass Spectrometry - Difference Gel Electrophoresis (DIGE): Methods and Protocols - page 52

3.2. In-Gel Protein
Digestion with Trypsin

1. Cover the gel pieces with suffi cient ACN:50 mM ABC in a 1:1
(v/v) ratio.
2. Shake the plate in a thermomixer for 10 min at 25°C.
3. Remove the destaining solution.
4. Repeat steps 1-3 until the pieces are clear (see Note 5).

3.2.1. Destaining ( see Note 4)

1. Add 100 L of ACN to the gel plugs.

2. Shake the plate in a thermomixer for 10 min at 25°C.
3. Discard the ACN.
4. Repeat steps 1-3.
5. Centrifuge the samples briefl y to ensure that all gel spots are at
the bottom (see Note 6).

μ 3.2.2. Dehydration

1. Add cold trypsin solution to the samples.
2. Incubate on ice for 20 min and remove the excess enzyme
solution.
3. Add 25 mM ABC to completely cover the gel plugs and incubate
the plate in a thermomixer over night at 36°C.

3.2.3. Digestion
( see Note 7)

First extraction :
1. Add 50

3.2.4. Extraction
of Peptides (see Note 8)

L of ACN on top of the buffer and shake the plate in
a thermomixer for 10 min at 25°C.
2. Remove the solutions from each well and transfer individually
to clean 0.6-mL Axygen tubes.

μ

Second extraction :
3. Add 30 L of ACN to each well and

shake the plate in a thermomixer for 10 min at 25°C.
4. Remove the solutions and combine them with the fi rst extraction
solutions.

μ L of pure water and 50 μ

Third extraction:
5. Add 50-100 L of ACN and shake the plate in a thermomixer

for 10 min 25°C.
6. Remove the peptide solutions and add them to the combined
extraction solutions.
7. Freeze the combined solutions for 2 h at −80°C and then
lyophilize.

μ