Titration to Determine the Minimum Amount of Protein to Stabilize the Gold

1. Adjust the pH of the gold sol to 0.5 pH units above the isoelectric point of the protein to be complexed. Care should be taken when adjusting the pH because nonstabilized colloidal gold will plug the pore of the pH electrode. Take an aliquot of a few milliliters of the gold and add five drops of 1% aqueous polyethylene glycol before measuring the pH. Make the necessary adjustments to the pH and repeat until the required pH is obtained. Do not return these aliquots to the remaining colloidal gold sol. Add 0.1 M HCl to lower the pH or add 0.2 M potassium carbonate to raise the pH, which each of these performed with vortexing.

2. Measure five aliquots of 0.5 mL of gold sol.

3. Prepare five aliquots of serially diluted protein in distilled water and add one of these, while shaking, to each of the 0.5-mL gold sol aliquots.

4. After 1 min, add 0.1 mL of 10% aqueous sodium chloride to each tube. Where there is excess protein in the tubes, the sol will not change color, but in those tubes where there is insufficient protein to stabilize the gold, flocculation will have occurred and the liquid will be blue. The correct concentration of protein is the minimal amount that will inhibit flocculation. Horisberger (10) suggests that for accurate determination of color change, a spectrophotometric assay should be used.

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