Blots gold labeling

The purified protein was used to raise polyclonal antibodies in a rabbit. The purified IgG showed significantly enhanced immunoreactivity towards a purified cytokinin oxidase preparation, when compared to non-immune control serum, by dot blotting using a gold labelled goat anti-rabbit second antibody procedure with silver enhancement. Most significantly, the antibody preparation was able to precipitate 80% of the cytokinin oxidase activity of a highly active, partially purified preparation from Zea mays kernels in the presence of fixed Staphylococcus aureus cells. No precipitation occurred with non-immune serum. 

Fig. 5. Immunodetection of cytokinin oxidase from Zea mays (lanes 1 and 2) and wheat seeds (lane 3) on a Western blot, using antibodies to Zea oxidase. Rabbit IgG was detected by gold-labelled goat anti-rabbit second antibody with silver enhancement...

The choice of label will depend on the application envisaged. Enzymes are widely applicable they are used in assays, such as ELISAs, and for detection of antigen blotted or dotted on membranes or embedded in tissue sections. Enzyme labels have also be used for the location of antigen in electron microscope sections but gold labelled antibodies (Chapter 11) are now extensively employed for this purpose. Antibodies labelled with a fluorescent molecule are used in assays and for the detection of antigens in tissue sections and also for flow cytometiy and fluorescence-activated cell sorting. 

Second antibodies may also be labeled with fluorescein isothiocyanate in which case the presence of an antigen is detectable by fluorescence upon irradiation of the blot with UV light. Sometimes, gold labeled or even biotinylated antibodies are used for the purpose of probing. 

Protein-labeled colloidal gold probes suit as well as for immuno-histochemistry in electron microscopy as for detection of antigens or glycoproteins on blots. There are several protocols for enhancement of electron density and visible color resulting in higher sensitivity. 

The detection of the antigen-antibody complexes on the membrane could be performed by a variety of techniques. The most common method is the application of a secondary antibody which binds specifically to the primary antigen-antibody complex. Hence, radiochemicals, flnorescent compounds, colloidal gold, enzymes, or biotin labels could be easily bound to the secondary antibody, and protein detection could be deduced from the intensity of the colored, fluorescent or chemiluminescent end product. Further details on blotting can be found elsewhere (Amersham, 1999). 

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