2. It is advisable to adjust this volume to the amount of tissue
analyzed in order to end up with a protein extract concentration closer to 5 rather than 1 or 10 μg/μL.
3. The number of ultrasonic cycles, time for pulse on and off and
amplitude need to be adjusted according to tissue-sample consistency and final volume of working solution, in order to
guaranty that sample is completely disaggregated and total
protein content released from cells. Working with lower
volumes (500 μL) of lysis buffer makes that during sonication
the solution heats more rapidly than working with higher
(1000 μL) volumes, increasing also the probability of formation of bubbles through cavitation process which would
decrease the sonication efficiency (if this happens, it would be
necessary to stop the procedure, and centrifuge the sample in
order to eliminate bubbles before resuming sonication
procedure).
4. For some tissues, a turbid layer is formed at the top of the
supernatant after centrifugation. The supernatant should be
carefully collected avoiding this layer.
5. At least for label-free quantitative proteomic approaches, it is
suggested that each sample (biological replicate) is run (and
analyzed by mass spectrometry) in triplicate (technical replicates). Reserve at least one lane for loading molecular weight
protein standards.
6. Electrophoresis time is highly dependent on specific user
needs. If extensive protein fractionation (high number of fractions from high to low MW) is required once SDS-PAGE has
been completed, then the running time could be a bit longer in
order to expand proteins a bit more along gel lane. Note that
under some circumstances when there is no interest in protein
fractionation, this procedure can also be convenient as it is used
for protein purification, an essential step before running protein digestion and mass spectrometry analysis.
7. Other protein staining methods compatible with MS analysis,
such as Coomassie blue, are equally valid after following longer
(fixation–staining–washing) protocols though in this same case
with lower sensitivity. On the other hand, the main
ä
Fig. 2 (continued) conditions and same multiplexing capacity. The procedures are equally valid for other
multiplexing capacities, such as 10-plex. Three biological conditions are represented as A, B, and C followed
by a number representing the biological replicate. Note that for method A, as one tag is used for the master
pool (Ref.), only five tags remain available per experiment, so in the example three biological replicates were
included per condition (nine samples in total), and one extra channel was used to include a technical replicate
(C3-tr). In method B, all six tags are available for samples, which in the example was used to include four
biological replicates for each biological condition (twelve samples in total)
96
Angel P. Diz and Paula Sa ´ nchez-Marı ´n
analyzed in order to end up with a protein extract concentration closer to 5 rather than 1 or 10 μg/μL.
3. The number of ultrasonic cycles, time for pulse on and off and
amplitude need to be adjusted according to tissue-sample consistency and final volume of working solution, in order to
guaranty that sample is completely disaggregated and total
protein content released from cells. Working with lower
volumes (500 μL) of lysis buffer makes that during sonication
the solution heats more rapidly than working with higher
(1000 μL) volumes, increasing also the probability of formation of bubbles through cavitation process which would
decrease the sonication efficiency (if this happens, it would be
necessary to stop the procedure, and centrifuge the sample in
order to eliminate bubbles before resuming sonication
procedure).
4. For some tissues, a turbid layer is formed at the top of the
supernatant after centrifugation. The supernatant should be
carefully collected avoiding this layer.
5. At least for label-free quantitative proteomic approaches, it is
suggested that each sample (biological replicate) is run (and
analyzed by mass spectrometry) in triplicate (technical replicates). Reserve at least one lane for loading molecular weight
protein standards.
6. Electrophoresis time is highly dependent on specific user
needs. If extensive protein fractionation (high number of fractions from high to low MW) is required once SDS-PAGE has
been completed, then the running time could be a bit longer in
order to expand proteins a bit more along gel lane. Note that
under some circumstances when there is no interest in protein
fractionation, this procedure can also be convenient as it is used
for protein purification, an essential step before running protein digestion and mass spectrometry analysis.
7. Other protein staining methods compatible with MS analysis,
such as Coomassie blue, are equally valid after following longer
(fixation–staining–washing) protocols though in this same case
with lower sensitivity. On the other hand, the main
ä
Fig. 2 (continued) conditions and same multiplexing capacity. The procedures are equally valid for other
multiplexing capacities, such as 10-plex. Three biological conditions are represented as A, B, and C followed
by a number representing the biological replicate. Note that for method A, as one tag is used for the master
pool (Ref.), only five tags remain available per experiment, so in the example three biological replicates were
included per condition (nine samples in total), and one extra channel was used to include a technical replicate
(C3-tr). In method B, all six tags are available for samples, which in the example was used to include four
biological replicates for each biological condition (twelve samples in total)
96
Angel P. Diz and Paula Sa ´ nchez-Marı ´n