Preparation of Protein A---Gold Complexes

Add to the boiling, rapidly mixing solution of chloroauric acid 3 ml of a 1% sodium citrate solution. 

Reflux for 30 min. The color of the suspension will change from a dark blue to a red as the monodisperse colloidal gold particles are formed. 

Any of the particle sols prepared above may be used to adsorb macromolecules to create gold probes. To concentrate the suspensions, the solutions may be filtered through a small-pore filter. Centrifugation also may be done. Each protein-gold complexation should be optimized for the proper amount of protein to add to maintain stability of the colloid. This can be done according to the method described in Section 1. 

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 0.1 M 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. 

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

---

Roth, J. (1982) The preparation of protein A-gold complexes with 3 nm and 15 nm gold particles and their use in labeling multiple antigens on ultra-thin sections. Histochem. J. 14,791-801. 

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)... 

Functionalization of nanorods with polyelectrolytes has been carried out by layer-by-layer deposition (92). First, CTAB-coated nanorods are prepared. Since these nanorods are positively charged, they can adsorb cationic and anionic poly electrolytes. Functionalization of nanorods with dyes is possible a fluorescent dye, 4-chloro-7-nitrobenzofurazan has been functionalized on the surface of Ti02 nanorods (93). Functionalization with a photoactive molecule such as ruthenium(II) tris(bipyridine) is also possible (94). A thiol derivative of the bipyridyl complex (Ru(bpy)3+-Cs-SH) in dodecane thiol is used for the functionalization of gold nanorods. Functionalization of block magnetic nanorods is very useful (95), for example, in the separation of proteins. Consider a triblock nanorod consisting of only two metals, Ni and Au. If the Au blocks are functionalized with a thiol (e.g. 11-amino-1 undecane thiol) followed by covalent attachment of nitrostreptavidin, then one can... 

---