Gold Enhancement
Silver enhancement has long been used by microscopists to better visualize gold labeling, especially when using small gold particles. Silver is specifically deposited around the gold particle, growing it in size. For TEM, the 1.4 nm Nanogold® can be enlarged to 10-20 nm for clear visiblility, even at low magnifications. Using an initial small gold immunoprobe allows better penetration into tissues (up to 40 microns!) and better labeling of antigens. Then silver enhancement makes everything clearly visible. The development time can be extended to deposit more silver and make the signal visible by bright field (or reflection) light microscopy. With a little more development time, the signal can be seen by the naked eye, and can be used with dot blots or Westerns.
Nanoprobes has now extended this technology by introducing a GOLD developer, "GoldEnhance". It works similarly to silver enhancement, but deposits gold around the initial gold particle.
Why GOLD? There are a number of advantages:
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may safely be used before osmium tetroxide staining (silver is dissolved by the oxidizing agent O4; gold is stable)
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lower backgrounds than silver in some cases; autonucleation minimal even after 1-2 hours
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for SEM, gold gives a much better backscatter signal than silver
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compatible with physiological buffers (silver precipitates with chloride ion, as in PBS buffer; gold does not)
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reaction is less pH senstitive than silver, and GoldEnhance is near neutral for best structural preservation of biological samples (many silver enhancers have a pH of 3-4) excellent shelf life
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low viscosity for easy and accurate mixing of components
Negative Staining
Negative stains are reagents which contain heavy atoms and do not crystallize upon drying, so that they provide a uniform electron-dense stain for electron microscopy. They are used to visualize the edges of protein complexes, macromolecules and cells in suspension. Unlike positive stains such as osmium tetroxide, negative stains do not obscure labeling of the biological structures themselves. Nanoprobes offers two unique, high-quality negative stains: NanoVan and Nano-W.
Conducting Polymers
Polypyrroles are used to form a thin molecular monolayer which is highly conductive (better than carbon); this is an ideal substrate for electron microscopy. To prepare the layer, unsubstituted pyrrole is mixed with one of the Nanoprobes surface active pyrroles (30DP or 30DOP) and applied to a water surface containing ferric chloride. The properties of the film are controlled using Langmuir-Blodgett techniques, and it is then used to coat grids for electron microscope observation.
Undecagold
Undecagold (Au11) is smaller than Nanogold®, with a core of 11 gold atoms only 0.8 nm in diameter. It is ideal for ultra-high-resolution EM work such as scanning transmission electron microscopy, or for resolving elements of large structures by TEM in conjunction with image processing. Undecagold has been used to see the biotin binding sites on avidin to 1 nm resolution by electron microscopy. It is prepared in a form with one reactive arm for cross-linking to a specific site on a target molecule, and is available with different reactivities for labeling different sites.
Note: Single undecagold clusters are not routinely visualized directly in the TEM. Undecagold may be seen upon image processing of protein helices and crystals, or visualized en masse if there is a bulk deposition such as staining of an organelle. Also, undecagold develops more slowly and with less final silver deposition than Nanogold®. Therefore, for many applications we recommend Nanogold®.
Features of Undecagold
- Ultra small 0.8 nm gold core.
- Use to prepare the smallest possible gold probes.
- Highest possible resolution.
- Site specific covalent labeling with choice of reactivities.
Custom Labeling and Custom Synthesis
We are currently able to offer custom labeling with Nanogold® or colloidal gold-labeled antibodies. Nanogold® labeling is restricted to the preparation of labeled Fab; fragments from F(ab')2 fragments, or the labeling of IgG molecules. While we will be glad to consider other requests, our time and resource to undertake such syntheses are limited. Please be advised also that new syntheses frequently require much more work than anticipated, and if a similar procedure has not been demonstrated before, may be better handled as a contract research project or collaboration.
Before requesting a custom synthesis quotation, we recommend that you consider our labeling reagents, which you may use to label a wide variety of molecules. Monomaleimido-Nanogold® reacts slectively with thiols (such as cysteines); Mono-Sulfo-NHS-Nanogold® labels primary alihatic amines (N-terminal or lysine residues) while Monoamino-Nanogold®M can be used with a variety of homo- or heterobifunctional cross-linkers, or directly to label RNA or glycoproteins.
Guidelines
Because the gold cluster labels developed by Nanoprobes, incorporated are attached by covalent cross-linking, they may be used to label almost any molecule with a suitably reactive functionality. We have already conjugated our probes to proteins, lectins, peptides, lipids, biotin and cytoskeletally active probes such as modified phalloidins. Although we are currently only able to complete custom syntheses of Nanogold® or colloidal gold-labeled antibodies, we are pleased to advise on the use of our labeling reagents to label other molecules. Please telephone, fax or E-mail us. Since our researchers are not necessarily familiar with your particular probe or application, we can often provide quotations and suggestions much more quickly if you provide some key information. The following data are particularly useful:
Molecular weight: All our labeling reactions use specific molar ratios of gold to probe. We need the molecular weight to calculate how much gold labeling reagent we will need, how much of the probe we will need to use for labeling, and also which product isolation protocol to use.
Optical density or UV/visible absorbtion data: The extinction coefficients of undecagold, Nanogold® and FluoroNanogold at specific wavelengths have been accurately determined, and these values allow us to determine how successfully your probe has been labeled. If you supply or refer us to specific values for your probe, we can provide you with the exact ratio of gold cluster label to probe in the product.
