Preparing the Probe

Radioactive DNA probes for Southerns and Northerns
The objective is to create a radioactive copy of a double-stranded DNA fragment. The process usually begins with a estriction fragment of a plasmid containing the gene of interest. The plasmid is digested with particular restriction enzymes and the digest is run on an agarose gel. Since a plasmid is usually less than 20 kbp long, this results in 2 to 10 DNA fragments of different lengths. If the restriction map of the plasmid is known, the desired band can be identified on the gel. The band is then cut out of the gel and the DNA is extracted from it. Because the bands are well separated by the gel, the isolated DNA is a pure population of identical double-stranded DNA fragments.

The DNA restriction fragment (template) is then labeled by random hexamer labeling:

1. The template DNA is denatured; the strands are separated by boiling.
2. A mixture of DNA hexamers (6 nucleotides of ssDNA) containing all possible sequences is added to the denatured template and allowed to base-pair. They pair at many sites along each strand of DNA.
3. DNA polymerase is added along with dATP, dGTP, dTTP, and radioactive dCTP. Usually, the phosphate bonded to the sugar (the a-phosphate, the one that is incorporated into the DNA strand) is synthesized from phosphorus-32 (32P), which is radioactive.
4. The mixture is boiled to separate the strands and is ready for hybridization.
   

This process is diagrammed below (labeled DNA shown in gray):

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 radioactive.

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.

Hybridization
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.

Washing
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.

Note
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 has bound.

Autoradiography
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.

Enzymatic Development
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.

Summary