Proteins colloidal gold

Taban, C. H., and Cathieni, M. M. 1995. Microwave-aided binding of colloidal gold-protein-sub-stance P. In Immunogold-Silver Staining Principles, Methods, and Applications (M. A. Hayat, ed.), pp. 183-195. CRC Press, Boca Raton, FL. 

The following sections discuss the preparation of colloidal gold suspensions of various particle sizes and their use in labeling proteins for detection purposes. Gold-labeled molecules and proteins are available from a number of manufacturers (Janssen, E-Y Labs, and Nanoprobes). 

Determine the minimum amount of protein A required to stabilize the colloidal gold sol being used. The colloidal suspension should be adjusted, if needed, with 0.1M K2CO3 to pH 6-7. Measure the pH of the sol using a gel-filled electrode. Determining the stabilization amount of protein A can be done according to the method described in Section 1, this chapter. 

Mix a stabilizing amount of protein A plus an additional 10 percent with the appropriate volume of colloidal gold. For example, Herbener (1989) mixed 10ml of a 14nm gold particle sol at pH 6.9 with 0.3 mg of protein A dissolved in 0.2 ml water. Mix well. 

Horisberger, M., and Clerc, M.F. (1985) Labeling of colloidal gold with protein A. Histochemistry 82, 219. 

Jemmerson, R., and Agre, M. (1987) Monoclonal antibodies to different epitopes on a cellsurface enzyme, human placental alkaline phosphatase, effect different patterns of labeling with protein A-colloidal gold. J. Histochem. Cytochem. 35, 1277-1284. 

Fig. 1. Diagram of an EM immunogold assay localizing a protein on plastic sections. The primary antibody binds to an exposed surface epitope of the embedded cells. The antibody is then visualized by binding a second antibody coupled to a colloidal gold particle. The electron-dense gold particle visibly marks the position of the bound antibodies when visualized with the electron microscope. 

Romano EL, Romano M. Staphyloccal protein A bound to colloidal gold a useful reagent to label antigen-antibody sites for electron microscopy. Immunochemistry 1977 14 711-715. 

Incubate with protein A/5 nm colloidal gold without dilution for 5 min. 

Ready-to-use blocking solutions containing normal inactivated sera or BSA for use with colloidal gold conjugates are available from Aurion (http //www. aurion.nl/). When labeling with protein A and protein G conjugates, this step can be omitted. 

Colloidal gold A suspension (or colloid) of submicrometer-sized particles of gold in a fluid, usually water. A colloidal gold conjugate consists of gold particles coated with a selected protein or macromolecule, such as an antibody, protein A or protein G. Because of their high electron density, the gold particles are visible in the electron microscope without further treatment. 

For a complete functional study of a biological pathway, it is often necessary to confirm the important protein interactions by in vivo experiments. This can be done by demonstrating protein localizations on a microscopic level, for instance, by tagging proteins with the green fluorescent protein or localizing them with antibodies and colloidal gold particles using an electron microscope. Additional, very specific biochemical experiments are often required to confirm the putative protein function. 

For proper identification of the proteins of interest in a blot, immunodetected proteins must be compared to the total protein pattern of the gel. This requires the indiscriminate staining of all the proteins in the blot. Colloidal gold stain is... 

In another application, an optical trap was used to manipulate cell-surface proteins (29). First, colloidal gold particles conjugated to monoclonal antibod-... 

General Method for Conjugating Proteins to Colloidal Gold... 

Perform a saturation isotherm to determine the protein gold ratio for each protein and each size colloidal gold (see Note 1). 

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