12. Staining solution: 25% (v/v) ethanol, 8% acetic acid (v/v), and
0.1% w/v Coomassie brilliant blue R-250 (see Note 25). Mix
250 mL ethanol absolute and 80 mL acetic acid glacial (100%
anhydrous) in a graduated cylinder. Add deionized H 2 O up to
a volume of 1 L. Dissolve then 1 g Coomassie brilliant blue
R-250 by stirring.
13. Destaining solution: 25% (v/v) ethanol, 8% acetic acid (v/v) in
H 2 O. Mix 250 mL ethanol absolute and 80 mL acetic acid
glacial (100% anhydrous) in a graduated cylinder. Add deionized H 2 O up to a volume of 1 L (see Note 26).
14. 1% Bromophenol-blue: Mix 0.1 g bromophenol-blue with
10 mL of deionized H 2 O. Store at 4
C.
15. 1% Agarose: mix 0.5 g agarose with 50 mL deionized H 2 O and
add 0.1 mL 1% bromophenol-blue solution.
16. Ettan™ DALTsix Large Vertical Electrophoresis System or
similar for 26 Â 20 cm gels (see Note 27).
17. MultiTemp™ III Thermostatic Circulator or similar.
18. 1.0 mm GE Ettan™ DALT low fluorescence Glass Set or
equivalent.
19. DALT six Gel-Caster or similar to prepare lab-cast gels (see
Note 28).
20. UVP BioDoc-It2 Gel Imaging System or equivalent imaging
system, provided with a 520-nm band-pass filter.
21. White Light Plate (see Note 29).
22. PDQuest software for 2D gel analysis or equivalent.
2.5 In-Gel Protein
Digestion
1. 80% AcN solution: mix 80 mL of AcN (LC–MS grade) and
20 mL of H 2 O (LC–MS grade).
2. Trypsin resuspension buffer: 50 mM acetic acid. Add 1.42 μL
of acetic acid glacial (100% anhydrous) to 498.58 μL of LC–
MS H 2 O.
3. Sequencing-grade trypsin: 100 μg in 100 μL of 50 mM acetic
acid glacial (100% anhydrous), 1 μg/μL stock (see Note 30).
4. 25 mM ammonium bicarbonate (ABC), pH 8.0: dissolve
19.8 mg ABC (!99.5%) in 10 mL of LC–MS H 2 O (see
Note 31).
5. 0.5% TFA, 50% AcN solution: mix 50 mL AcN (LC–MS
grade), 0.5 mL TFA, and add LC–MS H 2 O to a graduated
cylinder up to 100 mL.
6. Refrigerated microcentrifuge.
7. Ultrasonic bath.
8. Thermoblock or incubator set to 37
C.
Fluorescent 2DE-LC-MS for Carbonylome Analysis
233
0.1% w/v Coomassie brilliant blue R-250 (see Note 25). Mix
250 mL ethanol absolute and 80 mL acetic acid glacial (100%
anhydrous) in a graduated cylinder. Add deionized H 2 O up to
a volume of 1 L. Dissolve then 1 g Coomassie brilliant blue
R-250 by stirring.
13. Destaining solution: 25% (v/v) ethanol, 8% acetic acid (v/v) in
H 2 O. Mix 250 mL ethanol absolute and 80 mL acetic acid
glacial (100% anhydrous) in a graduated cylinder. Add deionized H 2 O up to a volume of 1 L (see Note 26).
14. 1% Bromophenol-blue: Mix 0.1 g bromophenol-blue with
10 mL of deionized H 2 O. Store at 4
C.
15. 1% Agarose: mix 0.5 g agarose with 50 mL deionized H 2 O and
add 0.1 mL 1% bromophenol-blue solution.
16. Ettan™ DALTsix Large Vertical Electrophoresis System or
similar for 26 Â 20 cm gels (see Note 27).
17. MultiTemp™ III Thermostatic Circulator or similar.
18. 1.0 mm GE Ettan™ DALT low fluorescence Glass Set or
equivalent.
19. DALT six Gel-Caster or similar to prepare lab-cast gels (see
Note 28).
20. UVP BioDoc-It2 Gel Imaging System or equivalent imaging
system, provided with a 520-nm band-pass filter.
21. White Light Plate (see Note 29).
22. PDQuest software for 2D gel analysis or equivalent.
2.5 In-Gel Protein
Digestion
1. 80% AcN solution: mix 80 mL of AcN (LC–MS grade) and
20 mL of H 2 O (LC–MS grade).
2. Trypsin resuspension buffer: 50 mM acetic acid. Add 1.42 μL
of acetic acid glacial (100% anhydrous) to 498.58 μL of LC–
MS H 2 O.
3. Sequencing-grade trypsin: 100 μg in 100 μL of 50 mM acetic
acid glacial (100% anhydrous), 1 μg/μL stock (see Note 30).
4. 25 mM ammonium bicarbonate (ABC), pH 8.0: dissolve
19.8 mg ABC (!99.5%) in 10 mL of LC–MS H 2 O (see
Note 31).
5. 0.5% TFA, 50% AcN solution: mix 50 mL AcN (LC–MS
grade), 0.5 mL TFA, and add LC–MS H 2 O to a graduated
cylinder up to 100 mL.
6. Refrigerated microcentrifuge.
7. Ultrasonic bath.
8. Thermoblock or incubator set to 37
C.
Fluorescent 2DE-LC-MS for Carbonylome Analysis
233