- Blot cell
- BA 83 0.2-mm pore nitrocellulose sheets
- Buffer, PBS-Tween 20
- Antigenic proteins, antibodies, and horseradish peroxidase labeled
- Run an electrophoretic separation of known antigenic proteins according
to the procedures in Exercises 1 and 2.
- Draw a line 0.5 cm from the top edge of an 8 × 10 cm nitrocellulose sheet
and soak it in blot buffer for about 5 minutes.
Nitrocellulose is both fragile and flammable and easily contaminated
during handling. Wear prewashed gloves.
When soaking the microcellulose, wet 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 tray.
- 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
- 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 up.
- 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 cannot be separated.
- Roll a glass rod across the surface of the nitrocellulose to remove any air
bubbles and ensure 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
- 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 V dc.
- 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
- 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 prestained
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 4 times with 100 mL of
PBS-Tween 20 for 5 minutes each on a rocking platform.
- Cut the blot into 0.5-cm strips.
- Inactivate sera containing positive- and negative-antibody controls to the
antigens under examination by treating them 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
- Wash the strips 4 times for 5 minutes each with 10-mL quantities of PBSTween
20. The first wash should be done at 50°C, but the last 3 may be
done at room temperature.
- Add 3 mL of horseradish peroxidase-labeled antiglobulin, optimally diluted
in PBS-Tween and incubate at room temperature for 1 hour with continuous
- Wash the strips 4 times for 5 minutes each with PBS-Tween 20, and 1
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.
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 have the bands react with an enzyme
substrate that can be detected calorimetrically.
As a rule, however, most peptides are denatured during electrophoresis,
and of course, nucleic acids have no enzyme activity. The methods employed
for identifying nonenzymatic proteins and nucleic acids have been termed
Western for immunoblotting of proteins, Southern for techniques using DNA
probes, and Northern when using RNA probes. The probes are radioactive
complementary 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 analogy.
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 sandwiched between 2 layers of filters,
2 foam pads (for support), and 2 layers of a stainless steel mesh. This entire
apparatus can be submerged in a buffer and transfer allowed to occur by
diffusion (yielding 2 blots, 1 on each filter), or can be arranged in an electroconvective
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 nonspecific entities. DNA can be probed, for example, with cDNA,
or even a specific messenger RNA, to identify the presence of the gene for that