3. Wear a mask when weighing acrylamide. Transfer the weighed
acrylamide to the glass beaker and mix on a magnetic stirrer.
Unpolymerized acrylamide is a neurotoxin and care should be
exercised to avoid skin contact.
4. The acrylamide solution can be stored at 4
C for 1 month.
Acrylamide hydrolyzes to acrylic acid and ammonia. The acrylamide mixture, buffer, and water can be prepared in large
batches, frozen in aliquots, and used indefinitely. However, in
our laboratory, we make the acrylamide solution fresh when we
cast gels.
5. In our laboratory, we prepare this fresh each time.
6. Handle and store TEMED under inert gas, air, and moisture
sensitive at room temperature.
7. Simple method of preparing running buffer: Prepare 10Â
native buffer (0.25 M Tris–HCl, 1.92 M glycine). Weigh
30.3 g Tris–HCl and 144 g glycine, mix, and make it to 1 L
with water. Dilute 100 mL of 10Â native buffer to 890 mL
with water and add 10 mL of 10% SDS. Store the native buffer
at 4
C.
8. SDS precipitates at 4
C. Therefore, the loading buffer needs to
be warmed prior to use.
9. To label the samples TMT 10plex Isobaric Mass Tag Labeling
Kit was used.
10. To identify taxonomically the larvae isolated from the body
cavity of herring, perform the RFLP-PCR reaction according
to Mattiucci et al. described above (see ref. 8). You can also
perform the taxonomic identification based on highly specific
amplification of the genomic DNA (isolated with the use of
Xpure ™ Cell & Tissue) fragment from the ITS1/ITS2 region
with the use of Anis Sensitive Sniper Real-Time PCR kit
according to the manufacturer’s protocol using real-time
PCR method. The kit is used to identify representatives of
nematodes from the Anisakidae family found in the Baltic and
North Atlantic (Anisakis simplex s.s., A. pegreffii, Pseudoterranova decipiens, P. krabbei, Contracaecum osculatum, and Hysterothylacium aduncum (a species of the Raphidascarididae
family)).
11. A total of ten samples (five biological replicates of each developmental stage) is a maximum number for TMT 10plex labeling kit. You can also prepare fewer biological replicates and
include into the analysis of technical replicates. During this
step, you can replace homogenizer by crushing larvae manually
with a sterile plastic pestle in a centrifuge tube. If you are
culturing L3 larvae to L4 stage, homogenize as indicated
above 5–10 larvae per sample. Use the same number of larvae
72
Robert Stryin ´ ski et al.
acrylamide to the glass beaker and mix on a magnetic stirrer.
Unpolymerized acrylamide is a neurotoxin and care should be
exercised to avoid skin contact.
4. The acrylamide solution can be stored at 4
C for 1 month.
Acrylamide hydrolyzes to acrylic acid and ammonia. The acrylamide mixture, buffer, and water can be prepared in large
batches, frozen in aliquots, and used indefinitely. However, in
our laboratory, we make the acrylamide solution fresh when we
cast gels.
5. In our laboratory, we prepare this fresh each time.
6. Handle and store TEMED under inert gas, air, and moisture
sensitive at room temperature.
7. Simple method of preparing running buffer: Prepare 10Â
native buffer (0.25 M Tris–HCl, 1.92 M glycine). Weigh
30.3 g Tris–HCl and 144 g glycine, mix, and make it to 1 L
with water. Dilute 100 mL of 10Â native buffer to 890 mL
with water and add 10 mL of 10% SDS. Store the native buffer
at 4
C.
8. SDS precipitates at 4
C. Therefore, the loading buffer needs to
be warmed prior to use.
9. To label the samples TMT 10plex Isobaric Mass Tag Labeling
Kit was used.
10. To identify taxonomically the larvae isolated from the body
cavity of herring, perform the RFLP-PCR reaction according
to Mattiucci et al. described above (see ref. 8). You can also
perform the taxonomic identification based on highly specific
amplification of the genomic DNA (isolated with the use of
Xpure ™ Cell & Tissue) fragment from the ITS1/ITS2 region
with the use of Anis Sensitive Sniper Real-Time PCR kit
according to the manufacturer’s protocol using real-time
PCR method. The kit is used to identify representatives of
nematodes from the Anisakidae family found in the Baltic and
North Atlantic (Anisakis simplex s.s., A. pegreffii, Pseudoterranova decipiens, P. krabbei, Contracaecum osculatum, and Hysterothylacium aduncum (a species of the Raphidascarididae
family)).
11. A total of ten samples (five biological replicates of each developmental stage) is a maximum number for TMT 10plex labeling kit. You can also prepare fewer biological replicates and
include into the analysis of technical replicates. During this
step, you can replace homogenizer by crushing larvae manually
with a sterile plastic pestle in a centrifuge tube. If you are
culturing L3 larvae to L4 stage, homogenize as indicated
above 5–10 larvae per sample. Use the same number of larvae
72
Robert Stryin ´ ski et al.