Protein Protocols Protein Protocols

Immunogold 393
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Protein Staining and Immunodetection Using Immunogold
Susan J. Fowler
Introduction
Probes labeled with colloidal gold were originally used as electron-dense markers
in electron microscopy (1–3) and as color markers in light microscopy (4). Their
application to immunoblotting was not examined until later (5–7). The combination
of gold-labeled antibodies and protein A was demonstrated to be suitable for the
visualization of specific antigens on Western blots and dot blots (5,6). When goldlabeled
antibodies are used as probes on immunoblots, the antigen–antibody interaction
is seen as a pinkish signal owing to the optical characteristics of colloidal gold
(5). Used on its own, the sensitivity of immunogold detection is equivalent to indirect
peroxidase methods, and hence, only suitable for situations where there are
higher levels of antigen. In addition, the signal produced is not permanent. In order
to overcome this problem and to allow the technique to be used for more demanding
applications, a way of amplifying the signal was subsequently developed using the
capacity of gold particles to catalyze the reduction of silver ions (8). This reaction
results in the growth of the gold particles by silver disposition. A stable dark brown
signal is produced on the blot, and sensitivity is increased 10-fold. The sensitivity
achieved using immunogold silver staining (IGSS) is similar to that obtained with
alkaline phosphatase using colorimetric detection and several times more sensitive
than 125I-labeled antibodies. However, unlike colorimetric detection, the result is
stable and not prone to fading, and the chemicals used present no hazards. In addition,
the signal-to-noise ratio of IGSS is usually very high, and there are none of the
handling or disposal problems that are associated with 125I-labeled antibodies.
The binding of the gold to antibodies is via electrostatic adsorption. It is influenced
by many factors, including particle size, ionic concentration, the amount of protein
added, and its molecular weight. Most importantly, it is pH dependent (9). An additional
feature is that the binding of the gold does not appear to alter the biological or
immunological properties of the protein to which is attached. The colloidal gold
particles used to label antibodies can be produced in different sizes ranging from
1 to 40 nm in diameter. For immunoblotting, Amersham Pharmacia Biotech Ltd.
(Amersham, UK) supplies AuroProbe BL plus antibodies labeled with 10 nm particles.
From: The Protein Protocols Handbook, 2nd Edition
Edited by: J. M. Walker © Humana Press Inc., Totowa, NJ
393