N A N O P R O B E S     E - N E W S

Vol. 5, No. 9          September 17, 2004

Which properties are most important in your conjugate?

1. Attachment site of the gold label;
2. Stoichiometry: number of gold labels per conjugate;
3. Separation from gold or from unlabeled biomolecule;
4. Yield of labeled product?

The priority given to these issues will determine which reagent you use, how you do the labeling reaction, and how you isolate the conjugate. The issues are discussed in detail below:

1. Attachment site: where is the best place for the gold label?

   Nanogold is available with several different reactivities, for labeling different chemical groups in your molecule, and if you have more than one of these groups, you will have a choice of sites to attach the label. Generally, you should

   • Pick a position well away from the target binding site, so that the gold does not obstruct native reactivity; for example, a hinge disulfide is a good place to label IgG.
   • Choose a site known to be reactive and accessible to a large labeling reagent. If in doubt, test labeling first with a fluorescent label or easily detected dye with the same reactivity.

2. Labeling stoichiometry: how many gold labels do you want to attach?

   Do you need:

   • Exactly one gold per conjugate biomolecule?
   • Multiple gold labels conjugated to a single large biomolecule?
   • Multiple smaller molecules linked to a single larger gold particle?

   If you want a only one gold per conjugate, label at a unique site - i.e. a chemical group that occurs only once in your molecule. Then, you can decide which ratio of gold to biomolecule to use based on ease of product separation. Sometimes you can selectively reduce a one disulfide (for example, an IgG hinge disulfide) while leaving others intact, to generate a unique thiol which may be labeled with Monomaleimido Nanogold, a primary aliphatic amine which may be labeled with Mono-Sulfo-NHS-Nanogold, or a carbohydrate that may be oxidized and labeled with Monoamino Nanogold.

   However, if you wish to attach multiple gold labels in each conjugate, use a group known to be present at multiple sites, then use excess gold. If you want to attach several of your molecules to one gold particle, use a polyfunctional gold: positively charged Nanogold contains multiple amines, and can be activated using an appropriate heterobifunctional cross-linker for multiple conjugation; alternatively, Negatively Charged Nanogold contains multiple carboxylates, and may be activated using 1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC)/Sulfo-NHS for linking multiple copies of amino-functionalized probes.

   If you have no unique functional group in your biological molecule but want 1 : 1 labeling, use a 1 : 1 reaction ratio or small excess of biomolecule to ensure that only one gold label is available for each biomolecule. Another exception is NTA-Ni(II)-Nanogold, which possesses multiple NTA-Ni(II) groups: if you are using this reagent to label a His-tagged probe or target, you can control conjugate stoichiometry by using an excess of the NTA-Ni(II)-Nanogold to ensure that only one his-tagged biomolecule binds each gold.

3. Purity: which impurities must be removed?

   Is the complete removal of one of the unreacted reagents critical for your experiment? For example, if you are using gold to quench molecular beacons, then even very small amounts of unconjugated fluorescently labeled oligonucleotide may compromise the fluorescence properties of your product, so you need to ensure that these are completely removed. If you are labeling in order to solve the structure of a protein or large protein complex, removal of all unconjugated gold may be critical; however, if you are making an antibody conjugate to label a cell surface antigen, the presence of small amounts of unattached gold may not be a problem since they can be easily washed off after staining.

   If it is critical that your product does not contain one of the unreacted starting materials, plan to use an excess of the other to ensure that the critical one reacts to completion, and no unreacted form remains after conjugation. For example, if you are labeling molecular beacons, use a 5-fold to 10-fold excess of gold even if they are smaller than the Nanogold, to ensure that all the oligonucleotide reacts.

4. Separation method

   How do you plan to separate the conjugate? In our experience, gel filtration liquid chromatography, which works by separating molecules by size, is the most effective and reliable method for separating Nanogold conjugates. Our product information and instructions are optimized for this method: this means that we recommend performing the conjugation reaction using an excess of the smaller of the two reagents, so that the larger one is limiting, and reacts completely. For example, we recommend using excess gold when labeling antibodies, but excess biomolecule if you are labeling a smaller peptide. It is easier to separate the unreacted smaller reagent by gel filtration, because of the greater relative size difference between it and the conjugate.

   However, if you know you will be using a different method - such as gel electrophoresis or reverse-phase chromatography - then check to see if a different labeling procedure or reaction stoichiometry makes it easier to separate your product. For example, if you plan to purify a smaller labeled oligonucleotide by gel electrophoresis and know that the labeled and unlabeled oligonucleotide run very close together, while the gold is well separated, use an excess of the gold, so that the oligonucleotide is the limiting reagent and completely labeled; the same applies with alternative chromatographic methods such as ion exchange or reverse-phase chromatography. Sometimes it is worth running control separations with small quantities of the unlabeled biomolecule and unconjugated gold first, so that you can identify and separate unreacted starting materials.

Notes:

• Labeling reactions are planned stoichiometrically - using the ratio of the number of moles of Nanogold (sold in units of 30 nanomoles or 6 nanomoles) to the number of moles of the molecule you plan to label.

• How much to get? Usually, it's best to plan to use an excess (2-fold for small size differences, 5 to 20-fold for large size differences) of the smaller of your reagents.

• Are you doing multiple conjugations, or need to try the reaction a couple of times to find the right conditions? If so, then purchase the multi-use packaging (5 X 6 nmol rather than 1 X 30 nmol). Once dissolved, Monomaleimido Nanogold and Mono-Sulfo-NHS-Nanogold are rapidly hydrolyzed in aqueous solution.

Separation Selection Guide

We have found that the following media work well for gel filtration of different Nanogold-labeled conjugates:

These are suggestions that have worked well in our laboratories. You may find it worthwhile to check our separation section in our Guide to Gold Cluster Labeling, or the Pharmacia gel filtration selection guide for other gel filtration media, or products from other manufacturers, before proceeding.

Reverse-phase separation of Nanogold-labeled oligonucleotides is discussed in a previous article.

Please let us know if you need advice or assistance on the best methods for your labeling reaction. We will be glad to advise.

More information:

• Guide to Gold Cluster Labeling
• Guide: Spectra and Labeling Calculation
• Catalog information - Nanogold labeling reagents
• Technical Help Nanogold labeling reagents
• Technical Help - NTA-Ni(II)-Nanogold
• References - Nanogold Labeling

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Enzyme Metallography: a New Stain for Light and Electron Microscopy

Because the metal is deposited from solution and accumulated at the target site, rather than being carried there in particulate form, we thought that this method might enable much better penetration and antigen access for electron microscopy immunolabeling as well. Larger, readily visualized metal deposits up to tens of nanometers in size may be deposited at target sites, assembled like a ship-in-a-bottle from metal ions in solution, using a probe no larger than an enzyme-labeled antibody fragment or streptavidin. The process, and some preliminary results, are shown below:

Left: The enzyme metallographic process; Right: (a) Brachiola algerae (microsporida associated with opportunistic infection in humans) reacted to an antibody to polar tube followed by HRP-labeled secondary and metallographic substrate, diluted tenfold. The sporoblasts show staining with similar distribution to known PT distribution; (b) control with primary antibody omitted.

Enzyme metallography promises increased sensitivity, cleaner signals with lower background, and the ability to label even hard-to-reach antigens much more easily than conventional particulate immunolabeling methods. For more information and to learn more about this process, please visit our web site and read our papers from Microscopy and Microanalysis 2002, describing its use in light microscopy, and from the recent 2004 meeting where we describe its potential for correlative light and electron microscopy, and how it can increase detection sensitivity in blots and membrane detection. We are continuing to develop and optimize this method, and hope to introduce the reagent as a commercial product in the near future. Check our home page for announcements.

Our web site includes a number of other applications, both novel new uses for our current products (product applications), and preliminary results obtained with novel reagents (research applications).

Reference: