- [Instructor] The purpose of western blotting
is to separate proteins on a gel
according to their molecular weight.
The proteins are then transferred onto a membrane
where they can be detected using antibodies.
Heat the samples at 95 degrees C
for five to 10 minutes in a sample buffer
containing a reducing agent
such as beta mercaptoethanol .
This results in linearized proteins
with a negative charge proportional to their size.
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Place a gel into the electrophoresis tank
and add a buffer
insuring the tops to the wells are covered.
The acrylamide percentage of the gel
being used depends on the molecular weight
of the target protein.
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Load a molecular weight marker into the first lane.
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Then load the samples into adjacent wells.
All the samples should contain equal amounts of protein.
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Once all the samples are loaded,
add running buffer.
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Place the lid onto the electrophoresis tank.
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Turn on the power supply
and set the voltage recommended by the manufacturer
of the gels and the gel tank.
You should be able to see bubbles
rising through the tank.
Run the gel until the dye front
has moved sufficiently down the gel.
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The next stage is to transfer the proteins
from the gel onto a membrane.
Membranes are usually made from nitrocellulose or PVDF.
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Remove the gel from the tank
and carefully release it from its plastic case.
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Cut off the wells and the gel foot
and place the gel into transfer buffer.
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Prepare the transfer stack
by sandwiching the membrane and gel
between filter paper and sponges.
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The membranes should be closest to the positive electrode
and the gel closest to the negative electrode.
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Use a small roller to remove any bubbles
between the gel and the membrane.
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Clamp the transfer case closed,
and submerge into a transfer tank
containing transfer buffer.
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Add water to the outer chamber to keep the system cool
and put on the lid.
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Turn on the power supply to begin protein transfer.
Time and voltage will require optimization,
so check the manufacturer's instructions for guidance.
Now that the proteins have migrated from the gel
onto the nitrocellulose membrane,
the protein of interest can be detected with an antibody.
The membrane can be removed from the cassette
and the molecular weight marker should now be visible.
If required, the transfer of proteins
can be confirmed by staining the membrane
with Ponceau S solution.
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To prevent non specific binding of the antibody,
the membrane needs to be blocked.
Pour blocking buffer onto the membrane
and agitate gently on a rocker.
Typically this is done using a solution of five percent milk
or bovine serum albumin, BSA, for two hours
at room temperature or overnight at four degrees C.
The time and type of blocking buffer
should be optimized so check the data sheet
of the primary antibody you'll intend to use, for details.
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After the membrane is blocked, remove the blocking buffer
and add the diluted primary antibody in the same solution.
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Incubate on a rocker as before.
Typically, primary antibody incubations
are for one hour at room temperature,
or overnight at four degrees C.
Antibody concentration and incubation time
will need to be optimized.
Refer to the antibody data sheet for guidance.
Pour off the primary antibody and rinse the membrane twice
in wash buffer.
Follow with one 15 minute wash, and three 10 minute washes
on a rocker.
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The wash buffer is usually tris buffered saline, TBS,
or phosphate buffered saline, PBS,
with 0.1% tween 20.
Pour off the wash buffer and incubate membrane
in conjugating secondary antibody,
which has been diluted in blocking buffer.
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Usually this is done for one hour
at room temperature.
But antibody concentration and incubation time
will need to be optimized.
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Pour off the secondary antibody
and wash the membrane as shown previously.
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There are several different systems for detection.
If the secondary antibody is conjugated to an enzyme,
incubate the membrane
in the appropriate substrate before imaging.
If the secondary antibodies are fluorescent conjugates
then you can move directly onto the imaging step.
Imaging can be carried out with X-ray film
or with a digital imaging system.
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Place the membrane into an imaging tray.
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Place the imaging tray into the imaging system.
Exposure times will most likely need to be optimized
in order to clearly detect the bands
relating to the proteins of interest.
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