Protein Protocols Protein Protocols

394 Fowler
Fig. 1. Principle of the indirect visualization of antigens using immunogold probes. The
primary antibodies bind to immobilized antigens on the blot and are in turn recognized by goldlabeled
secondary antibodies. The above illustration of the binding pattern of secondary antibodies
to gold particles represents the type of conjugate formed with gold particles of 10 nm or
larger. The exact configuration adopted by the antibodies on the gold particle is not known.
For each 10 nm gold particle, there will be several antibodies bound, and the probe can
be considered to be a gold particle coated with antibodies (see Fig. 1). For certain
applications 1-nm gold particles allow increased labeling efficiency and can give
greater sensitivity than larger particles (10). This may be owing in part to the larger
number of intensifiable gold particles/unit of antigen (11). However, when using antibodies
labeled with 1-nm gold particles, the small size of the gold particles means that
visualization of the antigen–antibody interaction can be achieved only using silver
enhancement.
Silver enhancement was first reported by Danscher (8), who used silver lactate and
hydroquinone at a pH of 3.5. In the presence of the gold particles, that act as catalysts,
the silver ions are reduced to metallic silver by the hydroquinone. The silver atoms
formed are deposited in layers on the gold surface, resulting in significantly larger
particles and a more intense macroscopic signal (see Fig. 2). This classical enhancement
worked well, but had several disadvantages. The system was sensitive to light
and chemical contamination. In addition, the components were not stable and were
prone to self-nucleation, a phenomenon whereby the reduction of silver ions occurs
spontaneously in solution to form silver particles that can be deposited and lead to high
background.
More recently, silver-enhanced reagents have been developed that overcome these
problems. IntenSE BL (Amersham Pharmacia Biotech) is light insensitive and has a
neutral pH. It also exhibits delayed self-nucleation, which allows a fairly large time
margin before it is necessary to stop the reaction (see Fig. 3). Using silver enhancement
allows sensitivity to be increased 10-fold over immunogold detection.
In addition to the immunological detection of proteins, gold particles can also be
used as a general stain for proteins on blots (12). AuroDye forte (Amersham Pharmacia
Biotech Ltd) is a stabilized colloidal gold solution adjusted to a pH of 3.0. At this pH,
the negatively charged gold particles bind very selectively to proteins by hydrophobic
and ionic interactions. The proteins thus stained appear as dark red. The sensitivity