Protein Protocols Protein Protocols

Reutilization of Western Blots 441
is treated sequentially with unlabeled primary antibodies and radiolabeled secondary
antibodies. Prior to drying, the blot is briefly incubated in a protein-containing buffer.
After subsequent drying and autoradiography, the antibodies are removed by treating
the nitrocellulose with SDS at 65–70°C under reducing conditions. Minor modifications
of this procedure permit removal of peroxidase-coupled antibodies after detection
of antigens by enhanced chemiluminescence. These procedures are described in detail
in Subheadings 2–4. of this chapter.
1.2. Reutilization of Blots Without Removal of Antibodies
The preceding techniques were developed in an era when radiolabeled antibodies or
lectins were widely utilized for blotting (16). At the present time, enzyme-coupled
secondary antibodies are more widely utilized for immunoblotting; and fluorochromecoupled
antibodies are also being investigated (6). The use of these nonradioactive
reagents raises the possibility of detecting multiple antigens by either simultaneous or
sequential probing of blots without intervening dissociation of bound antibodies.
When two antigens of interest can be readily distinguished by size, blots have been
probed with two different immunological reagents simultaneously. Bound primary
antibodies are then visualized by probing with one or more radiolabeled or enzymecoupled
secondary antibodies as needed. Interpretation of results obtained in this
manner can be difficult, however, if one or more of the immunological reagents
cross-reacts with multiple species on the blot or one of the polypeptides of interest
exists as multiple species, for example, splice variants or posttranslationally modified
forms. A recently described alternative to this procedure involves the use of primary
antisera raised in several different species followed species-specific secondary
antibodies coupled to fluorochromes with distinguishable spectral properties (6). This
approach has the advantage of allowing unequivocal detection of the species that react
with each primary antibody. Its disadvantages, however, include the need for primary
antisera raised in different species and the current lack of widespread availability of
suitable imaging equipment.
An alternative to simultaneous probing involves sequential probing of blots with
different reagents. Krajewski et al. (5) reported that results obtained during sequential
probing of blots with different primary antibodies and peroxidase-coupled secondary
antibodies could be simplified by incubating blots with chromogenic peroxidase
substrates after each chemiluminescent detection. In brief, the chromogenic substrates
irreversibly inhibit the peroxidase after chemiluminescent detection is complete,
insuring that the peroxidase bound to one secondary antibody is not active at the
time subsequent secondary antibodies are bound. We have recently observed that the
sodium azide added to blocking buffers as a preservative also inactivates horseradish
peroxidase. As a consequence, blots can be sequentially reacted with multiple different
secondary antibodies without interference provided that azide is present in the solution
containing the primary antibodies (26). This process is illustrated in Fig. 1, where the
same piece of nitrocellulose was sequentially reacted with immunological reagents
raised in chickens, rabbits, and mice. With the successive use of reagents raised in
different species, the signal obtained at each step is unaffected by the prior probings
(Fig. 1, lanes 1–3) even though the previously bound antibodies remain attached to the