6. Incubate 15 min under slow agitation in the dark.
7. Carefully replace the reducing solution by the alkylating one
(5–15 mL) using the same tube (see Note 56).
8. Incubate 15 min under slow agitation in the dark (see
Note 57).
9. Cut a paper electrode wick in half and add 15 μL of molecular
weight standards.
10. Remove any water excess from the gel surface using one filter
paper. Add a little amount of warmed 1% agarose solution, take
the already alkylated IPG gel and submerge the strip for a few
seconds in running buffer (anodic running buffer). Immediately put the strip on the top of the gel. Carefully push the strip
to the bottom by using a spatula, until the whole IPG gel lay in
direct contact with the surface of running gel (see Note 58).
11. Place the paper electrode wick soaked with the standards and
fill the empty space above the strip with more warmed 1%
agarose.
12. Repeat the process with all the strips.
13. Fill anode tank with an anodic running buffer to the first
marked fill line.
14. Place gels into the electrophoresis unit. Then, fill top cathode
tank with cathodic running buffer up to the second marked fill
line. Add more anodic running buffer to anode tank up to that
second marked fill line. Electrophoresis unit is coupled to
thermostatic circulator set at 15
C, which cools the system.
15. Run gels at constant amperage of 10 mA per gel (maximum
140 V and 20 W) for 1 h. Then, increase to 20 mA per gel
(maximum 240 V and 20 W) and run for about 15 h, or until
the dye front has reached the bottom of the gel. Then, continue running for 20 min more at same conditions to remove
the excess of FTSC free. Cover the system with aluminum foil
during the electrophoresis to protect gels from the light.
16. Remove gels and wash thoughtfully with distilled water (see
Note 59).
17. Place gel in a glass container with 1:1 ethanol/ethyl acetate (see
Note 60).
18. Leave glass container slowly shaking for 5 min.
19. Discard the ethanol/ethyl acetate and add deionized H 2 O.
20. Capture carbonyl fluorescence image on imaging system
provided with a 520-nm band-pass filter.
21. Wash gel 3Â in distilled water for 5 min.
22. Add Coomassie staining solution to cover gel and let it stain
overnight while gently shaking.
Fluorescent 2DE-LC-MS for Carbonylome Analysis
237
7. Carefully replace the reducing solution by the alkylating one
(5–15 mL) using the same tube (see Note 56).
8. Incubate 15 min under slow agitation in the dark (see
Note 57).
9. Cut a paper electrode wick in half and add 15 μL of molecular
weight standards.
10. Remove any water excess from the gel surface using one filter
paper. Add a little amount of warmed 1% agarose solution, take
the already alkylated IPG gel and submerge the strip for a few
seconds in running buffer (anodic running buffer). Immediately put the strip on the top of the gel. Carefully push the strip
to the bottom by using a spatula, until the whole IPG gel lay in
direct contact with the surface of running gel (see Note 58).
11. Place the paper electrode wick soaked with the standards and
fill the empty space above the strip with more warmed 1%
agarose.
12. Repeat the process with all the strips.
13. Fill anode tank with an anodic running buffer to the first
marked fill line.
14. Place gels into the electrophoresis unit. Then, fill top cathode
tank with cathodic running buffer up to the second marked fill
line. Add more anodic running buffer to anode tank up to that
second marked fill line. Electrophoresis unit is coupled to
thermostatic circulator set at 15
C, which cools the system.
15. Run gels at constant amperage of 10 mA per gel (maximum
140 V and 20 W) for 1 h. Then, increase to 20 mA per gel
(maximum 240 V and 20 W) and run for about 15 h, or until
the dye front has reached the bottom of the gel. Then, continue running for 20 min more at same conditions to remove
the excess of FTSC free. Cover the system with aluminum foil
during the electrophoresis to protect gels from the light.
16. Remove gels and wash thoughtfully with distilled water (see
Note 59).
17. Place gel in a glass container with 1:1 ethanol/ethyl acetate (see
Note 60).
18. Leave glass container slowly shaking for 5 min.
19. Discard the ethanol/ethyl acetate and add deionized H 2 O.
20. Capture carbonyl fluorescence image on imaging system
provided with a 520-nm band-pass filter.
21. Wash gel 3Â in distilled water for 5 min.
22. Add Coomassie staining solution to cover gel and let it stain
overnight while gently shaking.
Fluorescent 2DE-LC-MS for Carbonylome Analysis
237