27. Large-format gels allow highest resolution and maximum
protein load.
28. Precast 26 Â 20 cm gels are also commercially available.
29. White Light Plate which converts UV transillumination to
white light for viewing Coomassie Blue-stained gels.
30. Trypsin stock (1 μg/μL) can be kept frozen in aliquots at
À20
C.
31. Prepare prior to use.
32. Initial tissue amount and extraction volume can be adjusted
according to sample characteristics and devices available in the
laboratory (ultracentrifuge rotor and tubes). Keep in mind that
the 2D analysis of carbonylated proteins in large gels require a
relatively high amount of protein although strongly depends
on sample nature.
33. Add protease inhibitors following the commercial instructions.
For ProteoBlock™ Protease Inhibitor Cocktail (Thermo
Fisher Scientific Inc), add 10 μL per 1 mL of the buffer.
34. Homogenization must be done on ice to prevent warming the
sample.
35. We usually divide protein extracts in aliquots of 300 μL each
but it will depend on protein concentration.
36. We usually prepare reaction mixtures of 600 μg protein in
600 μL total volume, which is the maximum reaction volume
allowed to carry out the FTSC-labeling protocol in 1.5 mL
tubes.
37. Hydrazone bonds formed between FTSC and carbonyls are
stable enough for fluorescent labeling experiments and does
not require an additional reduction step with sodium borohydride (NaBH 4 ) or sodium cyanoborohydride (NaCNBH 3 ).
38. 20% TCA solution should be cold.
39. Precipitation is working when the solution becomes cloudy.
Small sample amount may require longer precipitation times.
40. Eliminate as much as possible excess TCA and free FTSC by
several washes with ethanol/ethyl acetate. However, those
ethanol/ethyl acetate wash steps should be done carefully;
otherwise, you could lose much sample. Work under the
fume hood.
41. Use a flattened pipette tip or sonication to break up the protein
pellet if necessary.
42. Persist with the washing until the color of 1:1 ethanol/ethyl
acetate remains transparent after vortexing.
43. Avoid overdrying the pellet. Alternatively, dry pellet on air
under the fume hood.
242
Lucı ´a Me ´ ndez et al.
protein load.
28. Precast 26 Â 20 cm gels are also commercially available.
29. White Light Plate which converts UV transillumination to
white light for viewing Coomassie Blue-stained gels.
30. Trypsin stock (1 μg/μL) can be kept frozen in aliquots at
À20
C.
31. Prepare prior to use.
32. Initial tissue amount and extraction volume can be adjusted
according to sample characteristics and devices available in the
laboratory (ultracentrifuge rotor and tubes). Keep in mind that
the 2D analysis of carbonylated proteins in large gels require a
relatively high amount of protein although strongly depends
on sample nature.
33. Add protease inhibitors following the commercial instructions.
For ProteoBlock™ Protease Inhibitor Cocktail (Thermo
Fisher Scientific Inc), add 10 μL per 1 mL of the buffer.
34. Homogenization must be done on ice to prevent warming the
sample.
35. We usually divide protein extracts in aliquots of 300 μL each
but it will depend on protein concentration.
36. We usually prepare reaction mixtures of 600 μg protein in
600 μL total volume, which is the maximum reaction volume
allowed to carry out the FTSC-labeling protocol in 1.5 mL
tubes.
37. Hydrazone bonds formed between FTSC and carbonyls are
stable enough for fluorescent labeling experiments and does
not require an additional reduction step with sodium borohydride (NaBH 4 ) or sodium cyanoborohydride (NaCNBH 3 ).
38. 20% TCA solution should be cold.
39. Precipitation is working when the solution becomes cloudy.
Small sample amount may require longer precipitation times.
40. Eliminate as much as possible excess TCA and free FTSC by
several washes with ethanol/ethyl acetate. However, those
ethanol/ethyl acetate wash steps should be done carefully;
otherwise, you could lose much sample. Work under the
fume hood.
41. Use a flattened pipette tip or sonication to break up the protein
pellet if necessary.
42. Persist with the washing until the color of 1:1 ethanol/ethyl
acetate remains transparent after vortexing.
43. Avoid overdrying the pellet. Alternatively, dry pellet on air
under the fume hood.
242
Lucı ´a Me ´ ndez et al.