in each sample. If you are able to get L4 developmental stage
from the natural host, e.g., from striped dolphin (Stenella
coeruleoalba) use one larva per sample because they are significantly larger than those from in vitro culture.
12. Reduce sample viscosity using a microtip sonicator.
13. Determine the concentration of each sample in triplicate. Use
samples at !1 mg/mL. Less concentrated samples may be
used; however, it might be necessary to use larger volumes of
sample, as well as of reagents.
14. Prepare three aliquots by 100 μg of each sample in case you
need to repeat the next steps. The rest of the supernatant freeze
at À80
C until the SDS-PAGE electrophoresis or in case to
repeat the analysis.
15. Calculate the volume of the methanol, chloroform, and water
by multiplying the amount of sample in μL (where was 100 μg
of protein), by the appropriate number of volumes of the
specified reagents, e.g., 3 volumes of water is 300 μL when
the sample was 100 μL (3 Â 100 μL).
16. The result is three layers: a large aqueous layer on top, a circular
flake of protein in the interphase, and a smaller chloroform
layer on the bottom.
17. After this step, samples could be preserved at À80
C until the
time of next steps.
18. This prevents contact with atmospheric oxygen (which inhibits
acrylamide polymerization) in addition to helping to level the
resolving gel solution.
19. Centrifuging the samples prior to the run helps remove insoluble debris, which could produce streaks in the protein lanes.
20. Gel may be kept in fixing solution overnight without affecting
stain performance.
21. SDS-PAGE electrophoresis is a control step to determine if the
protein extraction was done correctly. Additionally, reviewers
may want to see the stained gels with samples, which were
subjected to analysis.
22. Digestion should not exceed 16 h. Make the calculations and
start the protocol at the time proper for you (e.g., in the
evening to keep the digestion overnight).
23. Samples could be put to the locker/drawer. Tubes may also be
wrapped in the aluminum foil and this way kept on the laboratory table.
24. Maximal trypsin activity occurs at pH 7–9; the enzyme is
reversibly inactivated at pH < 4. If the pH of the sample is
too acidic/alkaline, add to the sample few but no more than
20 μL of 100 mM TEAB and check the pH again.
Shotgun Proteomics for Anisakis simplex
73
from the natural host, e.g., from striped dolphin (Stenella
coeruleoalba) use one larva per sample because they are significantly larger than those from in vitro culture.
12. Reduce sample viscosity using a microtip sonicator.
13. Determine the concentration of each sample in triplicate. Use
samples at !1 mg/mL. Less concentrated samples may be
used; however, it might be necessary to use larger volumes of
sample, as well as of reagents.
14. Prepare three aliquots by 100 μg of each sample in case you
need to repeat the next steps. The rest of the supernatant freeze
at À80
C until the SDS-PAGE electrophoresis or in case to
repeat the analysis.
15. Calculate the volume of the methanol, chloroform, and water
by multiplying the amount of sample in μL (where was 100 μg
of protein), by the appropriate number of volumes of the
specified reagents, e.g., 3 volumes of water is 300 μL when
the sample was 100 μL (3 Â 100 μL).
16. The result is three layers: a large aqueous layer on top, a circular
flake of protein in the interphase, and a smaller chloroform
layer on the bottom.
17. After this step, samples could be preserved at À80
C until the
time of next steps.
18. This prevents contact with atmospheric oxygen (which inhibits
acrylamide polymerization) in addition to helping to level the
resolving gel solution.
19. Centrifuging the samples prior to the run helps remove insoluble debris, which could produce streaks in the protein lanes.
20. Gel may be kept in fixing solution overnight without affecting
stain performance.
21. SDS-PAGE electrophoresis is a control step to determine if the
protein extraction was done correctly. Additionally, reviewers
may want to see the stained gels with samples, which were
subjected to analysis.
22. Digestion should not exceed 16 h. Make the calculations and
start the protocol at the time proper for you (e.g., in the
evening to keep the digestion overnight).
23. Samples could be put to the locker/drawer. Tubes may also be
wrapped in the aluminum foil and this way kept on the laboratory table.
24. Maximal trypsin activity occurs at pH 7–9; the enzyme is
reversibly inactivated at pH < 4. If the pH of the sample is
too acidic/alkaline, add to the sample few but no more than
20 μL of 100 mM TEAB and check the pH again.
Shotgun Proteomics for Anisakis simplex
73