2. Prepare the stacking gel by mixing 1.25 mL of stacking buffer,
0.67 mL of acrylamide mixture, and 3 mL water in a 50 mL
conical flask. Add 100 μL of SDS, 40 μL of APS, and 5 μL of
TEMED. Insert a 10-well gel comb immediately without
introducing air bubbles.
3. Prepare the protein standard according to the manufacturer’s
instructions.
4. Transfer 20 μg (use the calculations made earlier, see Subheading 3.2, step 5) of the protein from each sample (from the
supernatant preserved previously at À80
C; see Subheading
3.2, step 6).
5. Mix the protein samples with loading buffer in the ratio of 1:2
and heat for 5 min at 100
C.
6. Centrifuge the heated samples at 3000 Â g for 30 s to bring
down the condensate. Load the samples (10 μL of sample per
lane) and protein standard (in amount according to the manufacturer’s instructions) on the gel (see Note 19).
7. Electrophorese at 80 V until the sample has entered the gel and
then continue at 150 V till the dye front (from the bromophenol blue dye in the samples) reached the bottom of the gel.
8. Following electrophoresis, pry the gel plates open with the use
of a spatula. The gel remains on one of the glass plates. Rinse
the gel with water and transfer carefully to a container with
fixing solution (see Note 20).
9. Follow the steps of your own silver stain system for protein
detection in polyacrylamide gels (Fig. 5a; see Note 21).
3.4 Protein Digestion
(See Note 22)
1. Add 100 μL of 100 mM TEAB to the dried pellet of the sample
(Subheading 3.2, step 14).
2. Vortex and sonicate the sample on ice for 2 cycles of 30 s pulses.
Make sure that the pellet is completely dissolved. If necessary,
add up to 20 μL of 100 mM TEAB and repeat vortexing and
sonication.
3. Spin the sample. Set the tube shaker–thermoblock at 55
C.
4. Add 5 μL of 200 mM TCEP.
5. Vortex and spin the sample. Incubate 1 h at 55
C with gentle
agitation (400 rpm).
6. At the end of the incubation prepare the 375 mM IAA. Wrap in
the aluminium foil and protect from light.
7. Spin the sample after incubation. Set the tube shaker–thermoblock at 37
C.
8. Add to the sample 5 μL of IAA, gently vortex, and incubate
30 min at room temperature in the dark (see Note 23).
Shotgun Proteomics for Anisakis simplex
67
0.67 mL of acrylamide mixture, and 3 mL water in a 50 mL
conical flask. Add 100 μL of SDS, 40 μL of APS, and 5 μL of
TEMED. Insert a 10-well gel comb immediately without
introducing air bubbles.
3. Prepare the protein standard according to the manufacturer’s
instructions.
4. Transfer 20 μg (use the calculations made earlier, see Subheading 3.2, step 5) of the protein from each sample (from the
supernatant preserved previously at À80
C; see Subheading
3.2, step 6).
5. Mix the protein samples with loading buffer in the ratio of 1:2
and heat for 5 min at 100
C.
6. Centrifuge the heated samples at 3000 Â g for 30 s to bring
down the condensate. Load the samples (10 μL of sample per
lane) and protein standard (in amount according to the manufacturer’s instructions) on the gel (see Note 19).
7. Electrophorese at 80 V until the sample has entered the gel and
then continue at 150 V till the dye front (from the bromophenol blue dye in the samples) reached the bottom of the gel.
8. Following electrophoresis, pry the gel plates open with the use
of a spatula. The gel remains on one of the glass plates. Rinse
the gel with water and transfer carefully to a container with
fixing solution (see Note 20).
9. Follow the steps of your own silver stain system for protein
detection in polyacrylamide gels (Fig. 5a; see Note 21).
3.4 Protein Digestion
(See Note 22)
1. Add 100 μL of 100 mM TEAB to the dried pellet of the sample
(Subheading 3.2, step 14).
2. Vortex and sonicate the sample on ice for 2 cycles of 30 s pulses.
Make sure that the pellet is completely dissolved. If necessary,
add up to 20 μL of 100 mM TEAB and repeat vortexing and
sonication.
3. Spin the sample. Set the tube shaker–thermoblock at 55
C.
4. Add 5 μL of 200 mM TCEP.
5. Vortex and spin the sample. Incubate 1 h at 55
C with gentle
agitation (400 rpm).
6. At the end of the incubation prepare the 375 mM IAA. Wrap in
the aluminium foil and protect from light.
7. Spin the sample after incubation. Set the tube shaker–thermoblock at 37
C.
8. Add to the sample 5 μL of IAA, gently vortex, and incubate
30 min at room temperature in the dark (see Note 23).
Shotgun Proteomics for Anisakis simplex
67