Preparation of Colloidal Gold-Labeled Proteins

This section discusses the properties of gold particles as well as the common methods of labeling proteins and other biomolecules with them. The cited references should be consulted to obtain protocols for using these protein-gold complexes in assay and detection systems. 

Most preparations of colloidal gold consist of particles varying in diameter from about 5 nm to around 150 nm. The methods of forming small particle gold suspensions of known diameter are discussed in Section 2. 

An approximation of the correct amount of protein to be added to a gold sol to maintain stability of the colloid can be done using the following protocol (Slot and Geuze, 1984). 

After about 1 minute, add 0.25 ml of 10 percent NaCl to the gold/protein suspension. Mix well. 

The amount of protein added at the stabilization point plus 10 percent should be used to produce the final protein-gold conjugate. 

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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). 

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)... 

Although colloidal gold is readily conjugated to proteins, preparation of direct antibody/gold reagents is not often done. The simplest approach to labelling with colloidal gold is an indirect one, where the primary antibody is detected... 

As mentioned in 2.7 unlabeled or not fully labeled colloids are unstable, especially sensitive to electrolytes, therefore an efficient labeling strategy is needed The protein of interest is dissolved in 200 FI of water at 1 mg/ml. Serial dilutions (1 5 to 1 10) of the protein in distilled water are prepared with lOOFl of volume each. 500 FI of the pH-adjusted gold sol is added to each tube, and after 10 minutes 100 FI of 10% NaCl (electrolyte) in distilled water are added. Tubes with stabilized, means sufficiently covered colloids will maintain a red color, unstable gold sol will turn to violet and blue and finally flocculate. The second tube containing more protein than the one whose color changes to blue is sufficiently covered. With the optimal pH and amount for adsorption determined, the protein-colloid solution is prepared in the desired amount. Excess protein is removed by centrifugation, the pelleted colloid particles are re-suspended in a small volume to prepare more concentrated solutions. Often the use of protein concentrators (Centriprep spin columns) is preferred to remove protein and concentrate the colloids. 

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