This produces a radioactive single-stranded DNA copy of both strands of
the template for use as a probe.
Radioactive Antibodies for Westerns
Antibodies are raised by injecting a purified protein into an animal, usually a
rabbit or a mouse. This produces an immune response to that protein. Antibodies
isolated from the serum (blood) of that rabbit will bind to the protein used for
immunization. These antibodies are protein molecules and are not themselves
They are labeled by chemically modifying the side chains of tyrosines in
the antibody with iodine-125 (125I), which is radioactive. A set of enzymes
catalyzes the following reaction:
antibody-tyrosine + 125I– + H2O2 → H2O + 125 iodo-tyrosineantibody
Enzyme-conjugated Antibodies for Westerns
Antibodies against a particular protein are raised as above and labeled by
chemically cross-linking the antibody molecules to molecules of an enzyme. The
resulting antibody-enzyme conjugate is still able to bind to the target protein.
In all 3 blots, the labeled probe is added to the blocked filter in buffer and
incubated for several hours to allow the probe molecules to find their targets.
After hybrids have formed between the probe and target, it is necessary to
remove any probe that is on the filter that is not stuck to the target molecules.
Because the nitrocellulose is absorbent, some of the probe soaks into the filter
and must be removed. If it is not removed, the whole filter will be radioactive
and the specific hybrids will be undetectable.
To do this, the filter is rinsed repeatedly in several changes of buffer to
wash off any unhybridized probe.
In Southerns and Northerns, hybrids can form between molecules with similar
but not necessarily identical sequences (For example, the same gene from 2
different species). This property can be used to study genes from different
organisms or genes that are mutated. The washing conditions can be varied so
that hybrids with differing mismatch frequencies are maintained. This is called
“controlling the stringency”—the higher the wash temperature, the more stringent
the wash and the fewer mismatches per hybrid allowed.
Detecting the Probe-Target Hybrids
At this point, you have a sheet of nitrocellulose with spots of probe bound
wherever the probe molecules could form hybrids with their targets. The filter
now looks like a blank sheet of paper—you must now detect where the probe
If the probe is radioactive, the radioactive particles that it emits can expose
x-ray film. If you press the filter up against x-ray film and leave it in the dark
for a few minutes to a few weeks, the film will be exposed wherever the probe
bound to the filter. After development, there will be dark spots on the film
wherever the probe bound.
If an antibody-enzyme conjugate was used as a probe, this can be detected by
soaking the filter in a solution of a substrate for the enzyme. Usually, the
substrate produces an insoluble colored product (a chromogenic substrate) when
acted upon by the enzyme. This produces a deposit of colored product wherever
the probe bound.