9. Immediately place a comb, taking care not to introduce air
bubbles.
10. Allow the gel to polymerize (see Note 28).
11. Gels can be used immediately or stored at 4
C (see Note 29).
Perform gel loading and migration by following
steps 12–19.
12. Thaw protein standard at room temperature. It should not be
subjected to heat.
13. Thaw samples on ice (see Note 30).
14. Heat sample 10 min at 95
C.
15. Vortex in order to homogenize.
16. Spin centrifuge at room temperature (see Note 31).
17. In the meantime, prepare your gel for electrophoresis: place
your cassette into the electrophoresis tank, add migration
buffer until it covers the top of your gel and gently take out
comb (see Note 32).
18. Load your samples and the standard into the wells (see
Note 33).
19. Fix voltage at 90 V until the resolving gel has been reached,
then set at 150–170 V until the dye front reaches the bottom of
the gel.
Once the migration is terminated, immediately proceed
with protein transfer (see steps 20–29) onto nitrocellulose or
PVDF membrane (see Note 34).
20. Cut one nitrocellulose membrane and 4 Â Whatman paper a
little bit larger than the size of the gel.
21. In order to orient the membrane (see Note 35), label the
nitrocellulose membrane with an HB pencil or cut-out a corner
of the membrane.
22. Prepare the transfer sandwich by soaking the transfer cassette,
and the sponges in a bucket containing 1Â transfer buffer, and
start preparing the sandwich by placing the sponges and the
Whatman papers in this order, from the cathode to the anode:
black part of the cassette, corresponding to the cathode; two
sponges soaked in the transfer buffer; two Whatman papers
soaked in transfer buffer.
23. Once the migration is over, remove the gel and separate gel
plates with the use of a spatula. The gel will stick to one of the
plates.
24. Carefully remove the stacking gel with a spatula and all parts of
the gel that are not useful.
25. Pour a small volume of 1Â transfer buffer onto the gel, wet
your gloves (see Note 36) and carefully separate gel from the
plate and add to the sandwich (see Note 35).
364
Noe ´ mie Kempf et al.
bubbles.
10. Allow the gel to polymerize (see Note 28).
11. Gels can be used immediately or stored at 4
C (see Note 29).
Perform gel loading and migration by following
steps 12–19.
12. Thaw protein standard at room temperature. It should not be
subjected to heat.
13. Thaw samples on ice (see Note 30).
14. Heat sample 10 min at 95
C.
15. Vortex in order to homogenize.
16. Spin centrifuge at room temperature (see Note 31).
17. In the meantime, prepare your gel for electrophoresis: place
your cassette into the electrophoresis tank, add migration
buffer until it covers the top of your gel and gently take out
comb (see Note 32).
18. Load your samples and the standard into the wells (see
Note 33).
19. Fix voltage at 90 V until the resolving gel has been reached,
then set at 150–170 V until the dye front reaches the bottom of
the gel.
Once the migration is terminated, immediately proceed
with protein transfer (see steps 20–29) onto nitrocellulose or
PVDF membrane (see Note 34).
20. Cut one nitrocellulose membrane and 4 Â Whatman paper a
little bit larger than the size of the gel.
21. In order to orient the membrane (see Note 35), label the
nitrocellulose membrane with an HB pencil or cut-out a corner
of the membrane.
22. Prepare the transfer sandwich by soaking the transfer cassette,
and the sponges in a bucket containing 1Â transfer buffer, and
start preparing the sandwich by placing the sponges and the
Whatman papers in this order, from the cathode to the anode:
black part of the cassette, corresponding to the cathode; two
sponges soaked in the transfer buffer; two Whatman papers
soaked in transfer buffer.
23. Once the migration is over, remove the gel and separate gel
plates with the use of a spatula. The gel will stick to one of the
plates.
24. Carefully remove the stacking gel with a spatula and all parts of
the gel that are not useful.
25. Pour a small volume of 1Â transfer buffer onto the gel, wet
your gloves (see Note 36) and carefully separate gel from the
plate and add to the sandwich (see Note 35).
364
Noe ´ mie Kempf et al.