44. Keep in mind that FTSC-labeled samples must be protected
from light during the whole process.
45. Strip lengths and pH gradient should be chosen depending on
sample characteristics and experimental aims. Shorter strips
allow fast, cost-effective screening and are more suitable
when the most abundant proteins are of highest interest. Nevertheless, their sample load is limited. Longer strips render
maximal resolution and loading capacity but is much timeconsuming. Regarding pH range, use a broad-range pH strip
(pH 3–11 nonlinear) to overview total protein distribution in
the sample. That information might be used afterward for
design more targeted strategy.
46. Always wear gloves when handling IPG strips and the equipment that comes in contact with them. Thereby, you can
minimize protein contamination, which can result in artifactual
spots showing up in the resulting 2D spot patterns.
47. Wait until most of the sample volume soak the strip before
adding the mineral oil.
48. Volume of mineral oil depends on strip length (1–5 mL).
49. Once FTSC-labeled proteins are loaded onto the IPG gels,
strips must be protected from light.
50. The total monomer (acrylamide) concentration or % T can be
modified if necessary, according to the molecular weight of
carbonylated proteins in the sample. Usually the higher molecular weight, the less % T.
51. Notice that the volumes are calculated for six 1.0 mm thick,
26 Â 20 cm gels. Any change in number/size gels will require
to recalculate volumes.
52. Degasification of running gel solution is needed to eliminate
oxygen, which inhibits acrylamide polymerization.
53. Clean with ethanol all glasses and rest components of the gel
caster device before filling.
54. Maintain the gels wet during the storage at 4
C. For example,
introduce the gels into a bag and add some water (about
100 mL).
55. Reducing solution contains DTT, which ensures that disulfide
bridges on the proteins are broken.
56. Alkylating solution contains IAA, which alkylates thiol groups
on the proteins, preventing their reoxidation during electrophoresis, and thus decreasing streaking and other artifacts in
the second-dimension separation.
57. Both FTSC and IAA must be protected from light.
58. We usually cast six large running gels but twelve 11 cm IPG
strip. Large gel dimensions (26 Â 20 cm) allow us to fit two
Fluorescent 2DE-LC-MS for Carbonylome Analysis
243
from light during the whole process.
45. Strip lengths and pH gradient should be chosen depending on
sample characteristics and experimental aims. Shorter strips
allow fast, cost-effective screening and are more suitable
when the most abundant proteins are of highest interest. Nevertheless, their sample load is limited. Longer strips render
maximal resolution and loading capacity but is much timeconsuming. Regarding pH range, use a broad-range pH strip
(pH 3–11 nonlinear) to overview total protein distribution in
the sample. That information might be used afterward for
design more targeted strategy.
46. Always wear gloves when handling IPG strips and the equipment that comes in contact with them. Thereby, you can
minimize protein contamination, which can result in artifactual
spots showing up in the resulting 2D spot patterns.
47. Wait until most of the sample volume soak the strip before
adding the mineral oil.
48. Volume of mineral oil depends on strip length (1–5 mL).
49. Once FTSC-labeled proteins are loaded onto the IPG gels,
strips must be protected from light.
50. The total monomer (acrylamide) concentration or % T can be
modified if necessary, according to the molecular weight of
carbonylated proteins in the sample. Usually the higher molecular weight, the less % T.
51. Notice that the volumes are calculated for six 1.0 mm thick,
26 Â 20 cm gels. Any change in number/size gels will require
to recalculate volumes.
52. Degasification of running gel solution is needed to eliminate
oxygen, which inhibits acrylamide polymerization.
53. Clean with ethanol all glasses and rest components of the gel
caster device before filling.
54. Maintain the gels wet during the storage at 4
C. For example,
introduce the gels into a bag and add some water (about
100 mL).
55. Reducing solution contains DTT, which ensures that disulfide
bridges on the proteins are broken.
56. Alkylating solution contains IAA, which alkylates thiol groups
on the proteins, preventing their reoxidation during electrophoresis, and thus decreasing streaking and other artifacts in
the second-dimension separation.
57. Both FTSC and IAA must be protected from light.
58. We usually cast six large running gels but twelve 11 cm IPG
strip. Large gel dimensions (26 Â 20 cm) allow us to fit two
Fluorescent 2DE-LC-MS for Carbonylome Analysis
243