Comparative 2D DIGE Analysis of the Depleted Serum Proteome for Biomarker Discovery - Difference Gel Electrophoresis (DIGE): Methods and Protocols - page 218

gel plugs. Replace the silicone mat to prevent dust contamination
of the samples during incubation steps and stand for 5 min at
room temperature. Remove the solution and repeat.
4. Add 100 mL of 5 mM NH 4 HCO 3 and incubate for 10 min at
room temperature. Remove and replace with 200 mL of ace-
tonitrile. Incubate for 15 min at room temperature.
5. Remove the acetonitrile into an appropriate waste disposal con-
tainer. Allow the plugs to air-dry for 10 min at room tempera-
ture then rehydrate with 10 mL of trypsin working solution.
6. Allow the gel pieces to rehydrate for 15 min at room tempera-
ture. Add 10 mL of 5 mM NH 4 HCO 3 in 20% acetonitrile to
make up the total volume of each sample well to 20 mL con-
taining 100 ng of trypsin, 5 mM NH 4 HCO 3 , and 10% ace-
tonitrile (see Note 10). Replace the silicon sealing mat and
incubate the plate overnight at 37°C.
7. Following the overnight digestion, add 20 mL of 1% formic
acid to each sample well to inhibit the reaction. Place the plate
in a sonicating water bath such that only the bottom half of the
plate is submerged for 15 min.
8. Remove the liquid from each well and transfer to the Final Sample
Tube. If protein identifi cation is being performed by LC-MS, the
Final Sample Tube can be a labeled autosampler vial.
9. Add 50 mL of 50% acetonitrile containing 1% formic acid to
each well. Incubate the sample in a sonicating water bath for
15 min, remove the liquid, and transfer to the appropriate
Final Sample Tube.
10. Add 100 mL of 100% acetonitrile. Incubate the sample in a
sonicating water bath for 15 min, remove the liquid, and trans-
fer to the appropriate Final Sample Tube.
11. Dry down the peptide-containing extracts in the Final Sample
Tubes using a vacuum centrifuge until they are ~1 mL in vol-
ume. Reconstitute the samples to an appropriate volume for
LC-MS (between 5 and 8 mL, depending on the LC injection
volume) in 3% acetonitrile and 0.1% formic acid. Incubate the
sample in a sonicating water bath for 15 min to ensure the
peptides are fully solubilized. Store the samples at −20°C until
required. Centrifuge briefl y prior to LC-MS analysis.
4. Notes
1. Resuspend the lyophilized 25 nmol DIGE fl uor minimal Cy2,
Cy3, and Cy5 CyDyes in 125 mL of anhydrous N , N -
dimethylformamide to produce 200 pmol/mL stock solutions.
Aliquot the stocks into clearly labeled screw-top microfuge tubes
(1 mL), which should be fl ushed with argon before capping.