5. Determine the protein concentration of the supernatant using
established methods such as the BCA protein assay (see
Note 13).
6. Transfer 100 μg of the protein from each sample into a new
tube and preserve the rest of the sample at À80
C (see
Note 14).
7. Add three volumes of methanol and one volume of chloroform
and vortex the sample (see Note 15).
8. Add three volumes of water (see Note 15).
9. Centrifuge samples at 14,000 Â g for 5 min at 4
C (see
Note 16).
10. Remove the top aqueous layer carefully, without disturbing the
protein flake.
11. Add three volumes of methanol and vortex (see Note 15).
12. Centrifuge the samples at 14,000 Â g for 5 min at 4
C.
13. Remove as much methanol as it is possible.
14. Dry the pellet in a vacuum centrifuge (see Note 17).
3.3 SDSPolyacrylamide Gel
Electrophoresis
1. Mix 2.5 mL of resolving buffer, 3.33 mL of acrylamide mixture, and 4 mL water in a 50 mL conical flask. Add 100 μL of
SDS, 80 μL of APS, and 10 μL of TEMED and cast gel within a
7.25 cm  10 cm  1.5 mm gel cassette. Allow space for
stacking gel and gently overlay with water (see Note 18).
Fig. 4 The visualization of taxonomical identification of Anisakis larvae. Line M shows a molecular weight
standard. Lines 1–13 show patterns indicating Anisakis simplex s.s. species (250 bp, 620 bp)
66
Robert Stryin ´ ski et al.
established methods such as the BCA protein assay (see
Note 13).
6. Transfer 100 μg of the protein from each sample into a new
tube and preserve the rest of the sample at À80
C (see
Note 14).
7. Add three volumes of methanol and one volume of chloroform
and vortex the sample (see Note 15).
8. Add three volumes of water (see Note 15).
9. Centrifuge samples at 14,000 Â g for 5 min at 4
C (see
Note 16).
10. Remove the top aqueous layer carefully, without disturbing the
protein flake.
11. Add three volumes of methanol and vortex (see Note 15).
12. Centrifuge the samples at 14,000 Â g for 5 min at 4
C.
13. Remove as much methanol as it is possible.
14. Dry the pellet in a vacuum centrifuge (see Note 17).
3.3 SDSPolyacrylamide Gel
Electrophoresis
1. Mix 2.5 mL of resolving buffer, 3.33 mL of acrylamide mixture, and 4 mL water in a 50 mL conical flask. Add 100 μL of
SDS, 80 μL of APS, and 10 μL of TEMED and cast gel within a
7.25 cm  10 cm  1.5 mm gel cassette. Allow space for
stacking gel and gently overlay with water (see Note 18).
Fig. 4 The visualization of taxonomical identification of Anisakis larvae. Line M shows a molecular weight
standard. Lines 1–13 show patterns indicating Anisakis simplex s.s. species (250 bp, 620 bp)
66
Robert Stryin ´ ski et al.