Colloidal gold protein complexes

It has been reported that some commercial preparations of colloidal gold-antibody complexes may contain free active antibody. Such free antibody will compete with antibody-colloidal gold particles for antigen binding sites and may reduce labeling intensity. The presence of free protein may be identified using a simple test procedure (20)... 

The sections are floated on small drops of appropriately diluted primary antibody and intermittently microwaved for a total duration of 8 min 2 min with microwave oven on, 2 min with microwave oven off, 2 min with oven on, and 2 min with oven off. This is followed by washing twice with PBS for 15 sec in a microwave oven. The sections are treated outside the oven for 15 min with protein A-colloidal gold (10 nm) complex diluted 1 20 to 1 50 (determined by spectrophotometry) in PBS containing 1% BSA. They are washed in PBS followed by distilled water and counterstained outside the oven. 

Vernooy-Gerritsen, M. J.L.M. Leuinssen G.A. Veldink J.F.G. Vliegenthart. Intracellular localization of lipoxygenases-1 and -2 in germinating soybean seeds by indirect labeling with protein a-colloidal gold complexes. Plant Physiol. 1984, 76, 1070-1079. 

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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