pipette tip, or by using a Whatman paper. Be careful, as the gel
wells are, at this stage, highly sensitive to any movement.
33. We load 7 μL for the standard, and 7–12 μL for samples,
depending on the size of the well and the quantity of proteins
expected. Do not exceed half of a well volume as sample can
pass from one well to the other. In order to obtain nice vertical
migrations in all wells, do not leave any empty well, and rather
load them with 1Â Laemmli sample buffer with
β-mercaptoethanol.
34. The choice of the membrane type is personal and both work
well. We use nitrocellulose membranes. When using PVDF, the
use of methanol instead of ethanol is mandatory, leading to
more toxic waste to be treated.
35. If your loading is symmetrical, it is mandatory to give an
orientation to the membrane and to be extremely careful on
the orientation of the gel and the membrane while assembling
the sandwich. If it is asymmetrical, it is still recommended to
give an orientation to your membrane.
36. This will keep the gel from sticking to your gloves and will thus
minimize the risk of tearing.
37. Run 60 min for low molecular weight proteins such as H3 and
up to 90 min for high molecular weight proteins such as
E-cadherin and N-cadherin. The time will vary depending on
your proteins of interest and your power supply parameters.
38. Storage should be done between two sheets of Whatman paper
at À20
C after the gel has been dried out on a paper tissue at
room temperature. Do not over dry.
39. If ladder dyes are visible, it means that the proteins at this size
haven’t transferred entirely onto the membrane. In a next
experiment, leave the transfer longer but do not overrun the
transfer as small proteins can also pass through the membrane.
40. We perform incubations with 4–5 mL diluted antibody, within
little plastic bags sealed with a plastic sealer. The diluted antibody can be kept at À20
C for months for reuse. We use them
five times maximum or until the signal lowers.
41. We use secondary antibodies coupled to horseradish
peroxidase.
42. This step takes advantage of the property of peroxidase to
oxidize a peroxide solution.
43. It is important to quantify and ascertain the quality of the RNA
extraction before proceeding with the reverse transcription
because contaminants (proteins or solvent) could decrease
the efficiency of the reverse transcription and/or PCR. This
can simply be done with a spectrophotometer by measuring the
EMT Studied by Molecular Biology and DNA-FISH in Human Cells
379
wells are, at this stage, highly sensitive to any movement.
33. We load 7 μL for the standard, and 7–12 μL for samples,
depending on the size of the well and the quantity of proteins
expected. Do not exceed half of a well volume as sample can
pass from one well to the other. In order to obtain nice vertical
migrations in all wells, do not leave any empty well, and rather
load them with 1Â Laemmli sample buffer with
β-mercaptoethanol.
34. The choice of the membrane type is personal and both work
well. We use nitrocellulose membranes. When using PVDF, the
use of methanol instead of ethanol is mandatory, leading to
more toxic waste to be treated.
35. If your loading is symmetrical, it is mandatory to give an
orientation to the membrane and to be extremely careful on
the orientation of the gel and the membrane while assembling
the sandwich. If it is asymmetrical, it is still recommended to
give an orientation to your membrane.
36. This will keep the gel from sticking to your gloves and will thus
minimize the risk of tearing.
37. Run 60 min for low molecular weight proteins such as H3 and
up to 90 min for high molecular weight proteins such as
E-cadherin and N-cadherin. The time will vary depending on
your proteins of interest and your power supply parameters.
38. Storage should be done between two sheets of Whatman paper
at À20
C after the gel has been dried out on a paper tissue at
room temperature. Do not over dry.
39. If ladder dyes are visible, it means that the proteins at this size
haven’t transferred entirely onto the membrane. In a next
experiment, leave the transfer longer but do not overrun the
transfer as small proteins can also pass through the membrane.
40. We perform incubations with 4–5 mL diluted antibody, within
little plastic bags sealed with a plastic sealer. The diluted antibody can be kept at À20
C for months for reuse. We use them
five times maximum or until the signal lowers.
41. We use secondary antibodies coupled to horseradish
peroxidase.
42. This step takes advantage of the property of peroxidase to
oxidize a peroxide solution.
43. It is important to quantify and ascertain the quality of the RNA
extraction before proceeding with the reverse transcription
because contaminants (proteins or solvent) could decrease
the efficiency of the reverse transcription and/or PCR. This
can simply be done with a spectrophotometer by measuring the
EMT Studied by Molecular Biology and DNA-FISH in Human Cells
379