26. Add the membrane to the sandwich. Verify the orientation to
be able to identify the wells later.
27. Add two Whatman papers soaked into 1Â transfer buffer onto
the sandwich, carefully take out air bubbles by rolling over the
sandwich with a roller.
28. Finish the sandwich by adding two sponges soaked into 1Â
transfer buffer and closing the cassette, carefully taking care not
to introduce any air bubbles.
29. Insert the cassette into the system, immerse the system with 1Â
transfer buffer, transfer to an ice bucket filled up with ice (this
will avoid overheating of the cassette) and run in a cold room
60–90 min with the power supply set at 300 mA, 300 W and
300 V (see Note 37).
Once the transfer is completed proceed with protein visualization (see steps 30–35). This step will enable you to confirm the success of the transfer. The gel can be stored at À20
C
(see Note 38).
30. Remove the sandwich and verify that proteins have nicely
transferred onto the membrane (see Note 39).
31. Transfer the membrane to a small plastic container and wash it
in PBST for a few minutes.
32. Trash PBST and replace by Red Ponceau.
33. Slowly agitate on an orbital shaker until colored bands appear.
34. Rinse with demineralized water in order to see well-defined
protein bands.
35. Completely destain membrane by incubating it in PBST.
To prevent unspecific binding of the antibody to lower
background, perform membrane blocking (see step 36).
36. Incubate membrane 1 h in PBSTM at room temperature on an
orbital shaker.
Then, incubate the membrane with the primary and secondary antibodies (see steps 37–44).
37. Dilute primary antibodies in PBSTM as described in Table 1:
38. For antibodies against N-cadherin, E-cadherin, and occludin,
perform incubation overnight at 4
C on a turning wheel. For
antibodies against Vimentin and H3, incubate 1–2 h at room
temperature on a turning wheel (see Note 40).
39. Dilute your secondary antibody and incubate as described by
suppliers (see Note 41).
40. Once the incubation is terminated, recover the antibody for
reuse, and place your membrane in a small plastic box having
just the size of the membrane.
EMT Studied by Molecular Biology and DNA-FISH in Human Cells
365
be able to identify the wells later.
27. Add two Whatman papers soaked into 1Â transfer buffer onto
the sandwich, carefully take out air bubbles by rolling over the
sandwich with a roller.
28. Finish the sandwich by adding two sponges soaked into 1Â
transfer buffer and closing the cassette, carefully taking care not
to introduce any air bubbles.
29. Insert the cassette into the system, immerse the system with 1Â
transfer buffer, transfer to an ice bucket filled up with ice (this
will avoid overheating of the cassette) and run in a cold room
60–90 min with the power supply set at 300 mA, 300 W and
300 V (see Note 37).
Once the transfer is completed proceed with protein visualization (see steps 30–35). This step will enable you to confirm the success of the transfer. The gel can be stored at À20
C
(see Note 38).
30. Remove the sandwich and verify that proteins have nicely
transferred onto the membrane (see Note 39).
31. Transfer the membrane to a small plastic container and wash it
in PBST for a few minutes.
32. Trash PBST and replace by Red Ponceau.
33. Slowly agitate on an orbital shaker until colored bands appear.
34. Rinse with demineralized water in order to see well-defined
protein bands.
35. Completely destain membrane by incubating it in PBST.
To prevent unspecific binding of the antibody to lower
background, perform membrane blocking (see step 36).
36. Incubate membrane 1 h in PBSTM at room temperature on an
orbital shaker.
Then, incubate the membrane with the primary and secondary antibodies (see steps 37–44).
37. Dilute primary antibodies in PBSTM as described in Table 1:
38. For antibodies against N-cadherin, E-cadherin, and occludin,
perform incubation overnight at 4
C on a turning wheel. For
antibodies against Vimentin and H3, incubate 1–2 h at room
temperature on a turning wheel (see Note 40).
39. Dilute your secondary antibody and incubate as described by
suppliers (see Note 41).
40. Once the incubation is terminated, recover the antibody for
reuse, and place your membrane in a small plastic box having
just the size of the membrane.
EMT Studied by Molecular Biology and DNA-FISH in Human Cells
365