Updated: September 17, 2004

N A N O P R O B E S     E - N E W S

Vol. 5, No. 9          September 17, 2004


In this Issue:

This monthly newsletter is to inform you about techniques to improve your immunogold labeling, highlight interesting articles and novel applications of metal nanoparticles, and answer your questions. We hope you enjoy it and find it useful; as always, let us know if we can improve anything.

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Nanogold®: Plan your Labeling for Best Results

Before starting a labeling reaction, it's worth planning so that you have all the materials you need for each step and to make sure your reaction is optimized for the best results. The issues you should consider before you order the reagents are discussed below, followed by a reagent selection guide and separation selection guide:

What are your requirements for your conjugate?

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.

Reagent Selection Guide

Molecule to be labeled Labeling site(s) Nanogold reagent Other reagents you will need
Antibodies, Fab' fragments Hinge thiol
Amines, single or multiple
Monomaleimido Nanogold®
Mono-Sulfo-NHS-Nanogold®
Disulfide reduction: Dithiothreitol (DTT), mercaptoethylamine, or other reducing agent
Antibody Fab fragments Amines, single or multiple Mono-Sulfo-NHS-Nanogold® None
Proteins, peptides, other small molecules
(one per Nanogold)
Thiols, single or multiple
Amines, single or multiple
Monomaleimido Nanogold®
Mono-Sulfo-NHS-Nanogold®
Disulfide reduction: Dithiothreitol (DTT), mercaptoethylamine, or other reducing agent
Small proteins, peptides or other small molecules
(2 or more per Nanogold)
Amine Positively Charged Nanogold®
Negatively Charged Nanogold®
Positively Charged Nanogold: Homobifunctional amine-reactive cross-linker (bis-sulfo-succinimidyl suberate, BS3 or similar)
Negatively Charged Nanogold: activate with 1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC)/Sulfo-NHS or similar reagent.
Small proteins, peptides or other small molecules
(2 or more per Nanogold)
Thiol Positively Charged Nanogold® Heterobifunctional amine/thiol-reactive cross-linker (Sulfo-succinimidyl 4-N-maleimidomethyl cyclohexane-1-carboxylate, Sulfo-SMCC, or similar)
Small proteins, peptides or other small molecules
(2 or more per Nanogold)
Carboxyl Positively Charged Nanogold® 1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC)/Sulfo-NHS
Oligonucleotides Thiols
Amines
(Via modified phosphoramidites or enzymatic modification)
Monomaleimido Nanogold®
Mono-Sulfo-NHS-Nanogold®
None
Oligonucleotides 5'-phosphate
(tech help)
Monoamino Nanogold® 1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC)/Sulfo-NHS
RNA, Glycoproteins, other molecules cis-1,2-diols (sugars) Monoamino Nanogold®
(Application Note)
Sodium periodate; sodium cyanoborohydride or other non-thiol reducing agent-NHS
His-tagged Proteins or peptides Polyhistidine tags NTA-Ni(II)-Nanogold® None

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:

Biomolecule Molecular Weight Suggested Gels
Antibodies: whole IgG
Medium - large proteins
150,000
50,000 - 300,000
Superose-12 (Pharmacia)
Pre-packed columns or bulk media.
Antibodies: Fab' fragments
Small/medium proteins
50,000
10,000 - 50,000
Superdex-75 (Pharmacia)
Pre-packed columns or bulk media.
MW separation range: 500 - 70,000
Large proteins and protein complexes 200,000 and larger Superose-6 (Pharmacia)
Pre-packed columns or bulk media.
MW separation range: 5,000 - 1,000,000

A-5m (Bio-Rad)
Available in coarse, medium or fine grades.
MW Separation range: 10,000 - 5,000,000

Small proteins and peptides, oligonucleotides 7,000 and smaller Superdex-Peptide (Pharmacia)
Pre-packed columns or PG-30 bulk media.
MW separation range: 100 - 7,000
Substrate analogs and other small molecules 2,000 and smaller GH-25 desalting gel (contact Millipore)
Exclusion limit: 3,000 (desalting)

Sephadex G-25 (Pharmacia)
Hi-Trap columns or Bulk media.

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:

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Enzyme Metallography: a New Stain for Light and Electron Microscopy

At Microscopy and Microanalysis 2004, we presented some further results with our novel staining method, enzyme metallography. In this method, an enzyme targeted to a site of interest is used to deposit metal from solution. The reaction is very clean, with negligible background in many applications, and highly sensitive. Used as a detection method for in situ hybridization, for example, it will readily visualize individual gene copies, enabling the evaluation of gene amplification by conventional brightfield light 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:

[Enzyme Metallography: Mechanism and Electron Microscopy] (68k)]

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:

  • Furuya, F. R.; Joshi, V. N.; Hainfeld, J. F.; Powell, R. D., and Takvorian, P. M.: Enzymatic Metallography as a Correlative Light and Electron Microscopy Method. Microsc. Microanal., 10, (Suppl. 2: Proceedings) (Proceedings of Microscopy and Microanalysis 2004); Anderson, I. M.; Price, R.; Hall, E.; Clark, E., and McKernan, S., Eds.; Cambridge University Press, New York, NY, 1210CD (2004).

    More information:

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    Nanogold® Elucidates Targeting of Hepatitus C Core Protein

    Hepatitis C virus (HCV) is the major causative agent of non-A, non-B hepatitis. It is estimated to infect 3% of the worlds population; vial infection persists in about 80% of infected individuals, causing chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. The HCV core protein represents the first 191 amino acids of the viral precursor polyprotein and is cotranslationally inserted into the membrane of the endoplasmic reticulum (ER). Cleavage at position 179 by a recently identified intramembrane signal peptide peptidase generates a 179-amino-acid matured form of the core protein. Using confocal microscopy, a fraction of the mature core protein was found to be colocalized with mitochondrial markers in coreexpressing HeLa cells and in Huh-7 cells containing the full-length HCV replicon. Subcellular fractionation showed that this protein associates with purified mitochondrial fractions with no ER contaminants. The core protein also fractionated with mitochondrion-associated membranes, a site of physical contact between the ER and mitochondria.

    In combination with in vitro mitochondrial import assays, immunoelectron microscopy labeling with 5 nm gold and with Nanogold® was used to show that the core protein is targeted to the mitochondrial outer membrane. Lentivirally infected HeLa cells or Huh-7 replicon cells were grown on glass coverslips, fixed in 2.5% paraformaldehyde, and permeabilized with 0.05% Triton X-100 for 3 to 5 minutes. Anti-core antibodies (1:40) were added for 5 h, followed by a 6 hour incubation with 5nm gold-coupled anti-mouse secondary antibodies (HeLa cells) or 4 hours with Nanogold anti-mouse secondaries (Huh-7 cells) followed by washing and fixing in 2.5% glutaraldehyde (in sodium cacodylate buffer, pH 7.2; 15 minutes), enhancement with HQ Silver, and processing for electron microscopy.

    A stretch of 10 amino acids within the hydrophobic C terminus of the processed core protein, when fused to green fluorescent protein, conferred mitochondrial localization. The role of this mitochondrial targeting is not known at present, but the localization of the core protein in the outer mitochondrial membrane suggests that it could modulate apoptosis or lipid transfer, both associated with this subcellular compartment, during HCV infection.

    References:

    Schwer, B.; Ren S.; Pietschmann, T.; Kartenbeck, J.; Kaehlcke, K.; Bartenschlager, R.; Yen, T. S., and Ott M.: Targeting of hepatitis C virus core protein to mitochondria through a novel C-terminal localization motif. J. Virol., 78, 7958-7968. (2004).

    Further details of electron microscopy procedures:

    Kartenbeck, J.; Stukenbrok, H., and Helenius, A.: Endocytosis of simian virus 40 into the endoplasmic reticulum. J. Cell Biol., 109, 27212729 (1989).

    More information:

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    Gold-Based Fluorescence and Colorimetric DNA Detection

    Li and Rothberg shed further light on the interaction of gold particles with oligonucleotides, and describe simple and novel assays for detecting specific sequences, in papers in Analytical Chemistry and the Journal of the American Chemical Society.

    In the first paper, a simple fluorescence quenching assay was developed using 13 nm gold particles. These bind to single-stranded DNA, but not to double-stranded. A fluorescently labeled probe was added to a solution containing a complementary target; this solution was then mixed with a solution of the 13 nm gold particles. Fluorescence quenching was used to discriminate single-stranded (unbound) from double-stranded (bound) probe: the fluorescent probe hybridized to the target did not adsorb to the gold particle, while unhybridized probe was adsorbed, and fluorescence was quenched. Subfemtomole amounts of untagged target were detected in minutes using this procedure.

    Reference:

    Li, H., and Rothberg, L. J.: DNA sequence detection using selective fluorescence quenching of tagged oligonucleotide probes by gold nanoparticles. Anal. Chem., 76, 5414-5417 (2004).

    In the second paper, the authors exploit the color change that occurs when gold particles are aggregated. Usually, this occurs in the presence of salt; however, they find, surprisingly, that single-stranded DNA adsorbs to gold nanoparticles, with shorter sequences adsorbing more rapidly than longer ones, and protects them against aggregation.

    13 nm gold particles, prepared in the conventional manner by the sodium citrate reduction of tetrachloroauric acid, were used for the rapid colorimetric detection of PCR products. The gold particles were mixed with an annealed PCR product mixture, resulting in the adsorption of the annealed strands to the gold particles. Upon cooling, re-hybridization of the products resulted in aggregation of the gold particles and a color change from red to blue-gray; in the absence of complementary PCR products, the solution remained red. The assay is simple, and takes on the sensitivity of the PCR detection.

    Reference:

    Li, H., and Rothberg, L. J.: Label-free colorimetric detection of specific sequences in genomic DNA amplified by the polymerase chain reaction. J. Amer. Chem. Soc., 126, 10958-10961 (2004).

    Charge-based interaction of Nanogold® with DNA has also been described, using Positively Charged Nanogold to bind electrostatically; autometallographic enhancement of this construct provides a potential method for the preparation of molecular wires. The different binding modes that are possible through synthetic modification of the Nanogold surface may provide more options for the preparation of oligonucleotide constructs.

    More information:

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    Other Recent Publications

    Continuing the theme of DNA-gold nanostructures, Alivisatos and co-workers now report the preparation of gold-DNA-quantum dot nanostructures, as part of an investigation into whether conjugated gold particles can enhance the fluorescence properties of quantum dots in the same manner as gold surfaces. Streptavidin-linked quantum dots were reacted with biotinylated, single-stranded DNA, and the construct allowed to hybridize with gold-conjugated complementary strands; products were separated by gel electrophoresis, with different bands found to contain species with different numbers of gold particles per quantum dot.

    Reference:

    Fu, A.; Micheel, C. M.; Cha, J.; Chang, H.; Yang, H., and Alivisatos, A. P.: Discrete nanostructures of quantum dots/au with DNA. J. Amer. Chem. Soc., 126 10832-10833 (2004).

    As part of their studies of cellular changes underlying epilepsy, Tongiorgi and co-workers used HQ Silver to enhance 1 nm gold-labeled anti-digoxigenin for the in situ hybridization detection of target RNA sequences using digoxigenin-labeled riboprobes. Dendritic targeting of mRNA and local protein synthesis enable neurons to deliver proteins to specific postsynaptic sites; the authors demonstrated that epileptogenic stimuli induce a dramatic accumulation of Brain-Derived Neurotrophic Factor (BDNF) mRNA and protein in the dendrites of hippocampal neurons in vivo. Dendritic targeting of BDNF mRNA occurs when the cellular changes that underlie epilepsy are occurring, and is not seen after intense non-epileptogenic stimuli. MK801, an NMDA receptor antagonist that can prevent epileptogenesis but not acute seizures, prevents the dendritic accumulation of BDNF mRNA, indicating that dendritic targeting is mediated via NMDA receptor activation. These results suggest that dendritic accumulation of BDNF mRNA and protein play a critical role in the cellular changes leading to epilepsy.

    Reference:

    Tongiorgi, E.; Armellin, M.; Giulianini, P. G.; Bregola, G.; Zucchini, S.; Paradiso, B.; Steward, O.; Cattaneo, A., and Simonato, M.: Brain-derived neurotrophic factor mRNA and protein are targeted to discrete dendritic laminas by events that trigger epileptogenesis. J. Neurosci., 24, 6842-6852 (2004).

    Heering and co-workers confirm our previous report that enzyme-linked gold particles can "nanowire" the enzyme for accelerated turnover. Yeast iso-1-cytochrome c (YCC), chemisorbed on a bare gold electrode via Cys102, exhibits fast, reversible interfacial electron transfer ( k0 ) 1.8 v 103 s-1), demonstrated by cyclic voltammetry, and retains its native functionality. Vectorially immobilized YCC relays electrons to yeast cytochrome c peroxidase, and to both cytochrome cd1 nitrite reductase (NIR) and nitric oxide reductase from Paracoccus denitrificans, thereby revealing the mechanistic properties of these enzymes. On a microelectrode, nitrite turnover was measured for ~80 zmol (49 000 molecules) of NIR, coadsorbed on 0.65 amol (390,000 molecules) of YCC. This provides the basis for highly sensitive electrochemical detection methods.

    Reference:

    Heering, H. A.; Wiertz, F. G.; Dekker, C., and De Vries, S.: Direct Immobilization of Native Yeast Iso-1 Cytochrome c on Bare Gold: Fast Electron Relay to Redox Enzymes and Zeptomole Protein-Film Voltammetry. J. Amer. Chem. Soc., 126, 11103-11112 (2004).

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