Protein Staining and Immunodetection Using Immunogold

Abstract

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.