Mono-Sulfo-NHS-Undecagold In 5 aliquots

Product Information

UNDECAGOLD is the smallest gold label available, prepared using a discrete gold compound rather than a colloid. ¹This kit contains the UNDECAGOLD particle with a reactive sulfo-succinimide functionality incorporated into a ligand on the surface of the gold particle; this has a specific reactivity towards primary amines, and may be covalently linked to any protein bearing an accessible primary amine, as shown on the next page (figure 1). The reagent as supplied has been lyophilized from 0.1 M HEPES-NaOH at pH 7.5; dissolution in 1 ml deionized water will produce a solution of activated UNDECAGOLD in this buffer. UNDECAGOLD conjugates are intended for use in high resolution electron microscopy where the smallest possible probe and the lowest possible interference with the immunoreactivity of the conjugate are desired. They are stable to wide ranges of pH and ionic strength, and are not radioactive or carcinogenic. The labeling reagent should be stored at -20°C.

Figure 1: Schematic showing UNDECAGOLD labeling of a protein via reaction of an amine and a sulfo-succinimide group.

This product contains 50 nmol of activated undecagold reagent: this is sufficient to label up to 10 nmol of amine sites. This corresponds to 1.0 mg of a protein with a molecular weight of 100 000 and a single labeling site.

Thiol Caution

UNDECAGOLD particles degrade upon exposure to concentrated thiols such as beta-mercaptoethanol or dithiothreitol. If such reagents must be used, concentrations should be kept below 1 mM and exposure restricted to 10 minutes or less.

Labeling Proteins with Sulfo-Succinimido-Undecagold

For most proteins no pretreatment is necessary since amines will be present. The protein is reacted with the undecagold²in buffer solution at pH 7.5, either for 1 hour at room temperature or overnight at 4°C; the pH shopuld be adjusted to 7.5 after mixing. The UNDECAGOLD conjugated product may be isolated either by gel filtration, using a fine gel such as Pharmacia Superose 6 or 12, Superdex 75 or Amicon GCL-90. Alternatively, unbound UNDECAGOLD may be removed by dialysis. The recommended procedure is given below:

1. Dissolve the protein in 0.02 M sodium phosphate buffer at pH 7.4 with 150 mM sodium chloride (1 mL)
2. Dissolve the UNDECAGOLD reagent in 1 ml deionized water. Sufficient reagent is supplied to label 10 nmol of amine sites; if you are using a smaller amount, use a proportionately smaller amount of the UNDECAGOLD reagent (e.g. half the reagent supplied will label 0.75 mg of IgG). Once activated UNDECAGOLD is reconstituted with water it should be used immediately. The succinimide ester is hydrolyzed in aqueous solution).
3. Add the activated UNDECAGOLD solution to the reduced antibody, and raise the pH of the mixture carefully to 7.5 with dilute sodium hydroxide. Incubate for either 1 hour at room temperature, or 12-18 hours at 4°C.
4. Separate the unbound gold particles from the antibody conjugates using dialysis or gel exclusion chromatography.
   (a) DIALYSIS: Use a membrane with a molecular weight cutoff of 100,000 and as the exchange buffer use 0.02 M sodium phosphate at pH 7.4 with 150 mM sodium chloride; stir for 4-6 hours in 200 ml of buffer. Repeat twice with fresh buffer. The membrane will retain the UNDECAGOLD labeled antibody, but unbound UNDECAGOLD particles and any other low molecular weight impurities will pass through.
   (b) GEL FILTRATION: The UNDECAGOLD conjugate may be effectively isolated by HPLC using a gel such as Pharmacia Superose 6 or 12 (which fractionate a wide range of molecular weights) or Amicon GCL-90 (which excludes molecules of mass 30,000 or greater). Concentrate the reaction mixture to a suitably small volume using membrane centrifugation (e.g. Amicon Centricon-30 system). Elute with 0.02 M sodium phosphate at pH 7.4 with 150 mM sodium chloride; the first, pale yellow peak or shoulder is the conjugate, while the second, darker band is unbound UNDECAGOLD particles. For even higher purity, repeat the this process one more time.

The extent of labeling may be calculated from the UV/visible spectrum of the conjugate. UNDECAGOLD has the following extinction coefficients at specific wavelengths:

* Measured for 5 X 10^-6 M solution in methanol.

UNDECAGOLD conjugates should be stored in 0.02 M sodium phosphate buffer with 150 mM sodium chloride; if they are to be stored longer than three days, add 0.1 % bovine serum albumin and 0.05 % sodium azide to prevent bacterial contamination and to prevent the protein from adhering to the surfaces of the storage vessel.