2 Materials
Prepare all solutions using ultrapure water and analytical-grade
reagents. Prepare and store all reagents at room temperature
(unless indicated otherwise). Once prepared, reagents/kits could
be used in the next steps of the analysis. Due to that, do not throw
the solutions. Diligently follow all waste disposal regulations in
your laboratory when disposing of waste materials.
2.1 Laboratory
Equipment
1. Surgical scissors and tweezers.
2. Petri dish.
3. PCR thermocycler or real-time PCR thermocycler.
4. 6-Well culture plates.
5. Sterile incubator set for 37
C and 5% CO 2 in humid air for
in vitro culture.
6. Centrifuge tubes, 1.5 and 2 mL.
7. Low protein-binding centrifuge tubes, 2 mL.
8. Homogenizer.
9. Sonicator.
10. Centrifuge, minimum 14,000 Â g required.
11. Vortex.
12. Vacuum centrifuge.
13. Tube shaker–thermoblock.
14. HIFU device with a 1 mm probe tip.
15. Polyacrylamide gel electrophoresis system.
16. pH papers.
17. 96-Well microplate.
18. 96-Well microplate reader compatible with absorbance measurement at 480 and 562 nm.
19. Proxeon EASY-nLC II LC system coupled with LTQ-Orbitrap
Elite mass spectrometer or similar.
20. Set of pipettes and pipette tips.
21. Marker for tubes.
2.2 Parasites
Isolation and In Vitro
Culture
1. 0.9% Sodium chloride (NaCl): weight 0.9 g of NaCl and
transfer to a 100 mL glass beaker. Make up to 100 mL with
water. Store at 4
C.
2. Bactericidal and fungicidal solution: 100 mg of chloramphenicol, 80 mg of gentamicin sulfate, 0.625 mg of amphotericin B,
10,000 IU penicillin G, 10 mg of streptomycin sulfate, and
4.5 mL Hanks’ solution. Make up to 10 mL with 0.9% NaCl.
Store at 4
C.
Shotgun Proteomics for Anisakis simplex
61
Prepare all solutions using ultrapure water and analytical-grade
reagents. Prepare and store all reagents at room temperature
(unless indicated otherwise). Once prepared, reagents/kits could
be used in the next steps of the analysis. Due to that, do not throw
the solutions. Diligently follow all waste disposal regulations in
your laboratory when disposing of waste materials.
2.1 Laboratory
Equipment
1. Surgical scissors and tweezers.
2. Petri dish.
3. PCR thermocycler or real-time PCR thermocycler.
4. 6-Well culture plates.
5. Sterile incubator set for 37
C and 5% CO 2 in humid air for
in vitro culture.
6. Centrifuge tubes, 1.5 and 2 mL.
7. Low protein-binding centrifuge tubes, 2 mL.
8. Homogenizer.
9. Sonicator.
10. Centrifuge, minimum 14,000 Â g required.
11. Vortex.
12. Vacuum centrifuge.
13. Tube shaker–thermoblock.
14. HIFU device with a 1 mm probe tip.
15. Polyacrylamide gel electrophoresis system.
16. pH papers.
17. 96-Well microplate.
18. 96-Well microplate reader compatible with absorbance measurement at 480 and 562 nm.
19. Proxeon EASY-nLC II LC system coupled with LTQ-Orbitrap
Elite mass spectrometer or similar.
20. Set of pipettes and pipette tips.
21. Marker for tubes.
2.2 Parasites
Isolation and In Vitro
Culture
1. 0.9% Sodium chloride (NaCl): weight 0.9 g of NaCl and
transfer to a 100 mL glass beaker. Make up to 100 mL with
water. Store at 4
C.
2. Bactericidal and fungicidal solution: 100 mg of chloramphenicol, 80 mg of gentamicin sulfate, 0.625 mg of amphotericin B,
10,000 IU penicillin G, 10 mg of streptomycin sulfate, and
4.5 mL Hanks’ solution. Make up to 10 mL with 0.9% NaCl.
Store at 4
C.
Shotgun Proteomics for Anisakis simplex
61