11 cm IPG gels per one running gel without overlapping.
Thereby, we double the number of samples that we can run
in each experiment.
59. Avoid touching glasses as much as possible and change your
gloves often or wash them with ethanol.
60. Work always under the fume hood when handling the ethanol/
ethyl acetate solution. This step significantly reduces the background in the FTSC-labeled gel.
61. Ensure that all gels from the same experiment are destained the
same number of times. Do not delay this destaining step to
prevent sample variations and change in gel size which can
hinder spot matching. When the background is almost negligible, gels must be photographed as soon as possible since Coomassie gels will be analyzed to normalize carbonylation data.
To preserve the gels, dry them in 50% methanol solution to
maintain a permanent record. Alternatively, the “wet” gel can
be sealed in a plastic bag with a small amount of destaining
solution and stored for many months at 4
C.
62. PDQuest software measures spot quantity as the total intensity
of a defined spot, which corresponds to the amount of protein
in the actual spot in the gel. Therefore, in the FTSC-labeled
gels, protein carbonylation level is the total intensity of the
FTSC in the spot in arbitrary units. To normalize protein
carbonylation level with protein abundance, we calculate the
carbonylation index [7] by dividing the spot quantity in the
FTSC-labeled gel by the spot quantity (i.e., the total intensity
of the Coomassie in arbitrary units) in the corresponding
Coomassie-stained gel. It is especially important to the correct
overlapping of FTSC and Coomassie gels to achieve an accurate spot matching. Since almost always there are less carbonylated proteins than the total, spot matching can be a tricky step.
We recommend using molecular standards as a reference point.
It is really helpful to cut FTSC-labeled proteins directly under
UV exposition if it is possible to do it without any risk in your
UV transilluminator model. Then FTSC-labeled samples will
be digested and identified to confirm its identity. The gel
cutting under UV exposition will be also staining with Coomassie solution to check where the cut was done.
63. Use clean gloves and extreme the clean to avoid any external
contamination.
64. Cover completely the spot with the trypsin solution.
65. Check that the gel pieces are not dry and add more ABC buffer
if necessary.
66. Check the pH with an indicator paper. If pH is not acid (%2),
add a bit more 1% FA.
244
Lucı ´a Me ´ ndez et al.
Thereby, we double the number of samples that we can run
in each experiment.
59. Avoid touching glasses as much as possible and change your
gloves often or wash them with ethanol.
60. Work always under the fume hood when handling the ethanol/
ethyl acetate solution. This step significantly reduces the background in the FTSC-labeled gel.
61. Ensure that all gels from the same experiment are destained the
same number of times. Do not delay this destaining step to
prevent sample variations and change in gel size which can
hinder spot matching. When the background is almost negligible, gels must be photographed as soon as possible since Coomassie gels will be analyzed to normalize carbonylation data.
To preserve the gels, dry them in 50% methanol solution to
maintain a permanent record. Alternatively, the “wet” gel can
be sealed in a plastic bag with a small amount of destaining
solution and stored for many months at 4
C.
62. PDQuest software measures spot quantity as the total intensity
of a defined spot, which corresponds to the amount of protein
in the actual spot in the gel. Therefore, in the FTSC-labeled
gels, protein carbonylation level is the total intensity of the
FTSC in the spot in arbitrary units. To normalize protein
carbonylation level with protein abundance, we calculate the
carbonylation index [7] by dividing the spot quantity in the
FTSC-labeled gel by the spot quantity (i.e., the total intensity
of the Coomassie in arbitrary units) in the corresponding
Coomassie-stained gel. It is especially important to the correct
overlapping of FTSC and Coomassie gels to achieve an accurate spot matching. Since almost always there are less carbonylated proteins than the total, spot matching can be a tricky step.
We recommend using molecular standards as a reference point.
It is really helpful to cut FTSC-labeled proteins directly under
UV exposition if it is possible to do it without any risk in your
UV transilluminator model. Then FTSC-labeled samples will
be digested and identified to confirm its identity. The gel
cutting under UV exposition will be also staining with Coomassie solution to check where the cut was done.
63. Use clean gloves and extreme the clean to avoid any external
contamination.
64. Cover completely the spot with the trypsin solution.
65. Check that the gel pieces are not dry and add more ABC buffer
if necessary.
66. Check the pH with an indicator paper. If pH is not acid (%2),
add a bit more 1% FA.
244
Lucı ´a Me ´ ndez et al.