Exercise 4.8 - Western Blots
Figure 4.15 Samples of a Western blot
- Blot Cell
- BA 83 0.2 µ m pore nitrocellulose sheets
- Buffer, PBS-Tween 20
- Antigenic proteins, antibodies, and horseradish peroxidase
- Run an electrophoretic separation of known antigenic
proteins according to the procedures in Exercise 4.1
- Draw a line 0.5 cm from the top edge of an 8 x 10 cm
nitrocellulose sheet and soak it in blot buffer for about 5
Nitrocellulose is both fragile and flammable and easily
contaminated during handling. Wear gloves which are
When soaking the nicrocellulose wet first one side and then
turn the sheet over and wet the other, to prevent trapping
air within the filter.
- Place 200 ml of blot buffer into a tray and add a piece
of filter paper slightly larger than the electrophoretic gel
from Step 1.
- Remove the gel from the electrophoresis chamber after
the proteins have been separated, and place the gel into the
tray containing the filter paper. Do not allow the gel to
fall onto the paper, but place it next to the paper in the
- Gently slide the gel onto the top of the filter paper.
Keep the stacking gel off of the paper until the last
moment, since it tends to stick and make repositioning
- Holding the gel and the filter paper together, carefully
remove them from the tray of blot buffer and transfer the
paper and gel to a pad of the blot cell with the gel facing
- Transfer the nitrocellulose sheet (ink side down) onto
the top of the gel and line up the line drawn on the sheet
with the top of the stacking gel.
Once the gel and nitrocellulose touch they can not be
- Roll a glass rod across the surface of the
nitrocellulose to remove any air bubbles and insure good
contact between the gel and nitrocellulose.
- Lay another sheet of wet filter paper on top of the
nitrocellulose creating a sandwich of paper-gel-
nitrocellulose-paper, all lying on the pad of the blot cell.
- Add a second pad to the top of the sandwich and place
the entire group inside of the support frame of the blot
cell, and assemble the blot cell so that the nitrocellulose
side of the sandwich is toward the positive terminal.
- Check that the buffer levels are adequate and that the
cooling water bath is adjusted to at least 5° C. Subject
the gel to electrophoresis for 30 minutes with the electrodes
in the high field-intensity position. Follow the manufacturer
directions during this phase. Failure to closely monitor the
electrophoresis buffer or temperature can result in a fire.
Use a circulating cold bath appropriate to the apparatus and
hold the voltage to a constant 100 vdc.
- Upon completion of the electrophoresis (timed according
to manufacturer's directions), turn off the power and
disassemble the apparatus. Remove the blot pads from the
sandwich and remove the filter paper from the nitrocellulose
- Place the sandwich, nitrocellulose side down, onto a
glass plate and remove the other filter paper.
- Use a ball point pen to outline the edges of the
separating gel onto the nitrocellulose, including the
location of the wells. Carefully lift the gel away from the
nitrocellulose and mark the locations of the pre-stained
molecular weight standards as the gel is peeled away. Peel
the gel from the separating gel side, not the stacking gel.
- Wash the blot (the nitrocellulose sheet) at least four
times with 100 ml of PBS-Tween 20 for five minutes each on a
- Cut the blot into 0.5 cm strips.
- Inactivate sera containing positive and negative
antibody controls to the antigens under examination by
treating at 56 ° C for 30 minutes. Make dilutions
of 1:100 and 1:1000 of the controls with PBS-Tween 20.
- Place 3 ml of the diluted sera or controls onto a strip
from Step 16 and incubate for 1 hour at room temperature
while continuously rocking the sample.
- Wash the strips four times for 5 minutes each with 10 ml
quantities of PBS-Tween 20. The first wash should be done at
50° C but the last three may be done at room
- Add 3 ml of horseradish peroxidase-labeled antiglobulin,
optimally diluted in PBS-Tween and incubate at room
temperature for 1 hour with continuous agitation.
- Wash the strips four times for 5 minutes each with PBS-
Tween 20 and one more time with PBS only.
- Remove the PBS and add 5 ml of substrate solution.
Positive reaction bands usually appear within 10 minutes.
Stop the reaction by washing with water. Refer to Figure 4.15
for a comparison.
One of the more difficult tasks of electrophoretic
separations is the identification of specific bands or spots
within a developed gel. As observed with LDH isozymes, one
method of doing this is to react the bands with an enzyme
substrate that can be detected colorimetrically.
As a rule, however, most peptides are denatured during
electrophoresis, and, of course, nucleic acids have no
enzyme activity. The methods employed for identifying non-
enzymatic proteins and nucleic acids have been termed
Western for immunoblotting of proteins, Southern for
techniques using DNA probes Northern when using RNA
probes. The probes are radioactive complimentary strands of
nucleic acid. The first of these techniques was the
Southern, named for the developer of the procedure, Edward
Southern. Northern and then Western blots were named by
Blotting techniques first develop a primary gel:
protein on acrylamide; or DNA/RNA on agarose. The gel
patterns are then transferred to nitrocellulose membrane
filters and immobilized within the nitrocellulose membrane.
This process of transfer to an immobilizing substrate is
where the term blotting originated. The process is widely
used in today's laboratories because the immobilization
allows for extensive biochemical and immunological binding
assays that range from simple chemical composition to
affinity purification of monospecific antibodies and cell-
protein ligand interactions.
In practice, the electrophoresis gel is sandwhiched
between two layers of filters, two foam pads (for support)
and two layers of a stainless steel mesh. This entire
apparatus can be submerged in a buffer and transfer allowed
to occur by diffusion (yielding two blots, one on each
filter), or can be arranged in an electro-convective system
so that transfer occurs in a second electrophoretic field.
Once the transfer has occurred, the blots can be
probed with any number of specific or non-specific
entities. DNA can be probed, for example, with cDNA or even
a specific messenger RNA to identify the presence of the
gene for that message.
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Cell Biology Laboratory Manual
Dr. William H. Heidcamp, Biology Department, Gustavus Adolphus College,
St. Peter, MN 56082 -- firstname.lastname@example.org