Applications of Nanogold® and Related Reagents
Because of its site-specific covalent attachment to biomolecules, and its physical and chemical stability, Nanogold® lends itself to many applications for which colloidal gold is not suitable.
Product applications describe applications of our current products, while research applications describe novel probes and reagents which are not available as products or are under development as future new products.
Product Applications
Following are notes and reports on some unique results obtained using Nanogold® probes:
High-Density, Reliable Pre-Embedding Nanogold® Labeling Procedures
Procedure using HQ Silver Enhancement
- Higher density gold labeling than with other methods.
- Very high specificity.
- Maximum resolution.
This procedure has been described by Tanner and co-workers, and is reported to give significantly higher densities of silver-enhanced gold particles than other methods. An example of the results is shown below:
Materials and Reagents
- Sodium phosphate buffer: 0.1 M sodium phosphate, pH adjusted to 7.4.
- Phosphate-buffered saline (PBS) buffer: 0.02 M sodium phosphate buffer with 0.15 M sodium chloride, pH adjusted to 7.4.
- Phosphate-buffered saline (PBS) buffer: 0.02 M sodium phosphate buffer with 0.15 M sodium chloride, pH adjusted to 7.4, containing (a) 5 % bovine serum albumin and 0.05 to 0.1 % sodium azide; and (b) 1% goat serum and 0.1% NaN3 for 3-4 X 5 min
- Glutaraldehyde and paraformaldehyde.
- HQ Silver reagent (Nanoprobes).
- Deionized or distilled water.
Gold-Based Autometallography
Gold labels such as Nanogold®1 and colloidal gold2 are enlarged and visualized in the electron microscope or optically by the selective deposition of silver onto their surfaces. This process, known as autometallography (AMG), silver amplification3 or silver enhancement,4 is initiated by exposing the particles to a solution containing silver (I) ions and a reducing agent such as hydroquinone2,3 or n-propyl gallate.4 Particles may be enlarged to between 30 and 100 nm in diameter, giving a distinctive black, punctate staining in the light microscope. Nanogold® labeling with silver amplification is one of the most sensitive methods available for histopathology applications such as in situ hybridization. With Catalyzed Reporter Deposition (CARD; also called Tyramide Signal Amplification, or TSA®; NEN Life Sciences, Boston MA), it has been used to detect as few as 1-2 copies of viral DNA or RNA per cell.5 However, its uses are restricted by reactions of silver (I) with halides and other elements in tissues. Also, after signal development, self-nucleation and non-specific background deposition begin quickly, which can make end-point selection difficult or prevent incorporation into automated procedures.
We have found that gold can also be used as an autometallographic agent. In a suitable chemical environment, it may be selectively deposited onto Nanogold® or colloidal gold to generate a high contrast signal in the electron microscope, and black, punctate staining in the light microscope. It is also effective on blots. We find background staining to be equal to or lower than that found with silver enhancement in several test systems; in addition, although development is complete within 20 minutes, autonucleation is minimal even after two hours exposure to the reagent. This gives the gold autometallographic process a potential advantage for automation. In preliminary electron microscope experiments on the in situ detection of DNA and RNA and the labeling of CD44 in astrocytoma cells,6 particle sizes were found to be more uniform than those found using silver enhancement.
Using gold rather than silver has additional advantages. It can be safely used with osmium tetroxide and uranyl acetate staining, conditions which can etch silver-enhanced gold particles, without the need for protective gold toning. The gold autometallographic reaction is tolerant of a wider range of anions, and may be used in physiological buffers (even with chlorides, which precipitate silver; however, water washes are recommended). In the SEM, gold gives far superior backscatter detection compared with silver. Furthermore, the reaction is less pH sensitive than silver enhancement: the formulation is near neutral, which for some tissues is preferable to the low pH (~3.8) of many silver developers for morphological preservation. In in situ hybridization experiments, such as the detection of HPV-16 viral DNA in SiHa cells using CARD with Nanogold®-streptavidin detection, we find that gold autometallography to be as sensitive as silver amplification, with cleaner backgrounds (Figs. 1-4).
The gold autometallography reagent is prepared from three stable components mixed in equal volume, and applied to specimens for times from five minutes to twenty minutes. A "stop" treatment with 1-2 % sodium thiosulfate for 2 minutes was found to produce cleaner backgrounds in the in situ studies, although a simple water wash to stop development has also been used successfully.
Detection of Nanogold®-labeled molecules on Gels
Specific: Develops only Nanogold®-labeled band
Rapid: 1-5 minutes
Sensitive: More sensitive than usual gel stains
May be used directly on gels or on blot transfers
1.4 nm Nanogold® particles may be developed with silver so that they become visible to the naked eye, thus amplifying the signal thousands of times. If you have used monomaleimido-Nangold, mono-NHS-Nanogold® or mono amino-Nanogold® to label a protein or other molecule, these may then easily be analyzed and detected on gels using silver enhancement.
