9. Use IPG buffer according to the pH range of the IPG strip
selected.
10. Urea/Thiourea solution get colder while dissolving in water.
Leave it at room temperature to let it warm.
11. Urea solutions which are going to be stored should be treated
with a mixed-bed ion exchange resin, such as Serdolit MB-1, to
avoid the formation of cyanate, which causes protein carbamylation. That irreversible protein modification interferes with
isoelectrofocusing and MS analysis.
12. TCA is an irritant and corrosive and must be handled safely and
carefully.
13. Prepare only the convenient volume of ethanol/ethyl acetate
solution at the moment of use. Work always under the
fume hood.
14. To quantify samples resuspended in 2D buffer (urea/thiourea
buffer), Bradford is the most suitable assay. However, Bradford
reagent is not fully compatible with solutions at 6 M of urea.
We solve this problem by adding 2D buffer to the blank and all
standard curve step points.
15. Rehydration buffer is the 2D buffer described in step 4, Subheading 2.2.
16. Wear gloves and mask when handling SDS power because SDS
is an irritant and harmful by inhalation, ingestion, skin or eye
contact.
17. Dilute ultrapure glycerol solution to 50% to make pipetting
easier.
18. Prepare the convenient volume of reducing solution just before
using.
19. Prepare the convenient volume just before using. IAA solution
must be protected from light with aluminum foil.
20. APS solution can be stored at À20
C (no longer than
1 month) and never refreeze.
21. TEMED must be used under the fume hood to reduce odor.
22. The pH of this solution should not be adjusted.
23. Some electrophoresis units such as Ettan DALT from GE
HealthCare require the cathodic buffer of a higher concentration than the anodic buffer.
24. Any gel protein standards will work.
25. This Coomassie staining solution, which turns the entire gel
blue, simultaneously fixed and stained the proteins resolved on
the gel. Prepare under the fume hood.
26. Prepare under the fume hood.
Fluorescent 2DE-LC-MS for Carbonylome Analysis
241
selected.
10. Urea/Thiourea solution get colder while dissolving in water.
Leave it at room temperature to let it warm.
11. Urea solutions which are going to be stored should be treated
with a mixed-bed ion exchange resin, such as Serdolit MB-1, to
avoid the formation of cyanate, which causes protein carbamylation. That irreversible protein modification interferes with
isoelectrofocusing and MS analysis.
12. TCA is an irritant and corrosive and must be handled safely and
carefully.
13. Prepare only the convenient volume of ethanol/ethyl acetate
solution at the moment of use. Work always under the
fume hood.
14. To quantify samples resuspended in 2D buffer (urea/thiourea
buffer), Bradford is the most suitable assay. However, Bradford
reagent is not fully compatible with solutions at 6 M of urea.
We solve this problem by adding 2D buffer to the blank and all
standard curve step points.
15. Rehydration buffer is the 2D buffer described in step 4, Subheading 2.2.
16. Wear gloves and mask when handling SDS power because SDS
is an irritant and harmful by inhalation, ingestion, skin or eye
contact.
17. Dilute ultrapure glycerol solution to 50% to make pipetting
easier.
18. Prepare the convenient volume of reducing solution just before
using.
19. Prepare the convenient volume just before using. IAA solution
must be protected from light with aluminum foil.
20. APS solution can be stored at À20
C (no longer than
1 month) and never refreeze.
21. TEMED must be used under the fume hood to reduce odor.
22. The pH of this solution should not be adjusted.
23. Some electrophoresis units such as Ettan DALT from GE
HealthCare require the cathodic buffer of a higher concentration than the anodic buffer.
24. Any gel protein standards will work.
25. This Coomassie staining solution, which turns the entire gel
blue, simultaneously fixed and stained the proteins resolved on
the gel. Prepare under the fume hood.
26. Prepare under the fume hood.
Fluorescent 2DE-LC-MS for Carbonylome Analysis
241