N A N O P R O B E S     E - N E W S

Vol. 10, No. 4          April 30, 2009

Updated: April 30, 2009

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® with HQ Silver: Best Methods for Pre-Embedding EM Immunolabeling

Nanogold®-Fab' conjugates are the smallest commercially available immunogold probes, and their small size and high stability gives them several important advantages for pre-embedding immunolabeling:

  • High specimen penetration.
  • Highest resolution - the gold is nearer to the target.
  • High labeling density providing more quantitative labeling than other immunogold probes.
  • High level of access even to nuclear, restricted or hindered antigens.
Our HQ Silver enhancement reagent is formulated for best results with Nanogold: it is near neutral pH, with low ionic strength for minimal perturbation of morphology, and contains a natural thickening agent for uniform development and control of the rate of enhancement. There is an extensive literature on the use of these reagents for pre-embedding immunogold labeling.
Neuroscience is an area where this method has found particularly widespread application, and Tao-Cheng and colleagues - one of the groups that originally developed and optimized this method with Nanogold - used it to study the mechanism of synaptic spinule formation in brain tissue in their recent paper in Neuroscience. As a result, they established a relationship between synapse activity and spinule formation.

[Nanogold-Fab' size, STEM image, and pre-embedding labeling example (162k)]

Upper: Size comparison of Nanogold-Fab' with conventional 5 nm colloidal gold-IgG probe, showing overall probe size and distance of gold from target. lower left: Scanning transmission electron micrograph of Nanogold-labeled Fab', showing attachment of the Nanogold at the hinge region of the Fab' (image width 86 nm). lower right: Nanogold®-Fab' goat anti-rabbit IgG labeling the K+ channel Kv2.1 subunit in rat brain, followed by HQ Silver (Catalog # 2012) enhancement. Note high density and specificity of immunostaining, even elucidating subunit localization to cytoplasmic side of cell membrane and outer stacks of the Golgi; axons and terminals are clearly negative. Work done by J. Du, J.-H. Tao-Cheng, P. Zerfas, and C. J. McBain, NIH. See Neuroscience, 84, 37-48 (1998). Bar = 1 micron.

Spinules found in brain consist of small invaginations of plasma membranes, which enclose evaginations from adjacent cells. The authors used depolarization to study the dynamic properties of synaptic spinules: these are the most common type, found in synaptic terminals. To test whether depolarization triggers synaptic spinule formation, hippocampal slice cultures from 7-day-old rats, after 1014 days in culture, were subjected to depolarization treatment of 90 mM KCl (NaCl concentration correspondingly reduced) for 0.5, 1, 1.5, 2, 3, or 5 minutes. N-methyl-D-aspartic acid (NMDA) treatment (50 µM) was applied for 0.5, 1, 2, 3, 5 or 15 minutes; some slices were treated for up to 15 min with 250 µM of NMDA. To examine recovery after depolarization, high K+ medium was removed and the culture dishes washed three to four times in normal incubation medium for a total of 1, 2, 3, 5, 10, 30 and 60 minutes. In some experiments, recovery after high K+ treatment was performed in ice-cold incubation medium on ice. Experimental controls were processed in parallel, including all the medium changes and washing steps. Some slice cultures were fixed immediately without any treatment to assess their basal state.

For electron microscopy, slice cultures were fixed with 2% glutaraldehyde and 2% paraformaldehyde, or 4% glutaraldehyde in 0.1 N cacodylate buffer at pH 7.4 for 13 hours at room temperature, then stored at 4°C for 15 days. Acute brain slices were either fixed in the same manner, or directly with 1% osmium tetroxide (OsO4) in 0.1 N cacodylate buffer on ice for 1 hour, washed, and stored in buffer at 4°C. The fixed slice cultures were brushed off the insert filter, then in parallel with the acute slices, carefully trimmed for sampling from the CA1 region; slices were sectioned in the plane that retained their top and bottom surfaces for orientation. Aldehyde-fixed samples were postfixed with 1% OsO4 in 0.1 N cacodylate buffer for 1 hour on ice. All chemical-fixed samples (aldehyde and direct OsO4-fixed) were mordanted en bloc with 0.25% uranyl acetate in acetate buffer at pH 5.0 overnight at 4°C, washed, dehydrated through graded series of ethanol, and finally embedded in epoxy resins.

Immunogold labeling was conducted on samples fixed with 4% paraformaldehyde in PBS for 45 minutes to 1 hour, permeabilized with 0.1% saponin, and immunolabeled with clathrin antibody (clone X22). They were then incubated with secondary Nanogold-Fab' conjugate, and enhanced using HQ Silver, and finally embedded in epoxy resin for thin sectioning. Controls for immunolabeling included omission of primary antibody, and comparison with other, non-clathrin primary antibodies.

Virtually no synaptic spinules were found in control slices in the basal state, but numerous spinules appeared at both excitatory and inhibitory synapses after treatment with high K+. Spinule formation peaked with ~1 minute treatment at 37°C, then decreased with more prolonged treatment, and disappeared after 12 minutes of washout in normal medium. The rate of disappearance of spinules was substantially slower at 4°C. N-methyl-D-aspartic acid (NMDA) treatment also induced synaptic spinule formation, but less so than high K+ depolarization. In acute brain slices prepared from adult mice, synaptic spinules were abundant immediately after dissection at 4°C, extremely rare in slices allowed to recover at 28°C, but frequent after high K+ depolarization. High pressure freezing of acute brain slices followed by freeze-substitution showed that synaptic spinules are not induced by chemical fixation. This indicated that spinules are absent in synapses at low levels of activity, but form and disappear quickly during sustained synaptic activity. The rapid turnover of synaptic spinules may be linked to membrane retrieval during synaptic activity.

Reference:

  • Tao-Cheng, J. H.; Dosemeci, A.; Gallant, P. E.; Miller, S.; Galbraith, J. A.; Winters, C. A.; Azzam, R., and Reese, T. S.: Rapid turnover of spinules at synaptic terminals. Neuroscience, 160, 42-50 (2009).

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    Nanogold® Reagents; Stability and Solubility

    We are frequently asked about the stability of Nanogold® reagents in solution, as well as the solubility of Nanogold® in different solvents and how best to label hydrophobic or organic-soluble molecules such as peptides, lipids, or drugs.

    Because it is a coordination complex and not a colloidal gold particle, Nanogold is more stable than colloidal gold in aqueous buffers. It is not precipitated by salt, and can tolerate a wide range of pH from about 3 to 10, and salt concentration conditions. Nanogold is soluble in water and in most aqueous buffers, in most cases to at least 1,000 nmol/mL, providing a solution that is concentrated enough for most labeling experiments. However, solution may require gentle agitation for a short time before the reagent dissolves completely. However, Nanogold reagents are susceptible to the following conditions:

    • Activated reactive groups used for specific reactivity in the Nanogold labeling reagents Monomaleimido Nanogold and Mono-Sulfo-NHS-Nanogold are quickly hydrolyzed in aqueous buffers: therefore, it is important to mix the reconstituted Nanogold solution with the molecule to be labeled immediately.

    • Thiol caution: thiols have a very high affinity for gold surfaces: even low concentrations of thiol-containing reagent such as mercaptoethanol can quickly degrade Nanogold. Exposure should be restricted to 1 mM or lower concentrations for 10 minutes or less.

    However, the Nanogold particle itself is stable in aqueous solution; Nanogold reagents that do not contain activated functional groups - for example, Monomamino Nanogold or Positively Charged Nanogold can be stored in aqueous solution for up to several weeks provided they are not exposed to heat.

    Care should be taken to avoid including buffers or other molecules that might compete for labeling with the conjugate biomolecule. For example, you should avoid using TRIS buffer with Mono-Sulfo-NHS-Nanogold since it contains an amine that will react; likewise, avoid using cysteine in labeling reactions with Monomaleimido Nanogold.

    [Nanogold Labeling Reagents and Reactions (37k)]

    Monomaleimido Nanogold and Mono-Sulfo-NHS-Nanogold labeling reagents and labeling reactions. Reactivity occurs only through the reactive groups: the remainder of the Nanogold particle is coated with solubilizing ligands and does not adsorb to biological materials.

    Nanogold is also soluble in a number of organic solvents, including those that are well-tolerated by biological molecules. If necessary, including a proportion of one of these in the reaction mixture can increase solution of the reactive species and improve labeling and conjugate recovery:

    • Nanogold is soluble in alcohols, especially ethanol, although it is more soluble in alcohol-water mixtures than in alcohols alone. If you are using ethanol precipitation for a Nanogold-labeled oligonucleotide, then degree to which the labeled oligonucleotide will be precipitated will vary depending on the labeling stoichiometry and the length (and hence contribution to the solubility properties of the conjugate) of the oligonucleotide. Ethanol precipitation alone may give inadequate separation of Nanogold-labeled oligonucleotides, so we recommend that chromatographic separation (reverse-phase, gel filtration and hydrophobic interaction) and UV/visible spectroscopy are used in addition to ethanol precipitation to separate and characterize the reaction products in each phase of the reaction mixture. For a more detailed discussion of oligonucleotide labeling, see our archived article.

    • Nanogold is highly soluble in dimethylsulfoxide (DMSO): However, mixing DMSO with water liberates heat, and unless this mixing is undertaken very carefully, the heat can be detrimental to the Nanogold. Up to 20% DMSO is usually well tolerated by many biological molecules, and dissolving both the Nanogold reagent and molecule to be labeled in 20% DMSO-reaction buffer, cooled to room temperature, may improve labeling and recovery of insoluble or hydrophobic species such as some peptides or small organic molecules.

    • Nanogold is usually compatible with N,N-dimethylformamide (DMF): however, in many applications, we recommend the use of N,N-dimethylacetamide (DMA), which has very similar properties, instead, since Nanogold is soluble and more stable in this solvent. Nanogold is also soluble in acetonitrile.

    • If you are labeling lipids or other hydrophobic entities, Nanogold is soluble in mixtures of up to 50% dichloromethane (methylene chloride) or trichloromethane (Chloroform) with alcohols, and these should sufficiently solubilize both lipids and Nanogold to allow reaction. Addition of a small amount of an organic-soluble base, such as triethylamine, may help the reaction of Mono-Sulfo-NHS-Nanogold under these conditions but make sure that your base does not contain primary or secondary amines that may react with the Nanogold reagent.

    • Triethylammonium bicarbonate is an excellent solvent for Nanogold. It is prepared by bubbling carbon dioxide through a mixture of degassed triethylamine and degassed water; as the carbon dioxide dissolves and the mixture hydrates, the weakly ionic solution is formed. You should prepare a 0.6 M solution in 20% isopropanol; make a more concentrated solution in water first, then dilute in water / isopropanol as appropriate. More details are given in the following reference:

      Safer, D.; Bolinger, L., and Leigh, J. S.: Undecagold clusters for site-specific labeling of biological macromolecules: simplified preparation and model applications. J. Inorg. Biochem., 26, 77-91 (1986).

    Solvent is also a consideration in labeling surfaces. Here, the effect that the solvent-surface interaction has on the ability of the Nanogold to access the surface group must be considered. If you use a solvent that does not wet the surface sufficiently (such as aqueous buffers with hydrophobic polymer surfaces) the gold particles may be hindered from approaching the surface, and adding an organic solvent such as DMSO to enhance wetting will facilitate reaction.

    More information:

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    Nano-W and 2-Dimensional Infrared Show Protein Amyloid Formation Pathway

    Protein aggregation and crystallization phenomena, such as those in prion disease processes, provide a useful application for Negative stains, such as our NanoVan (methylamine vanadate) and Nano-W (methylamine tungstate): one of the most effective characterization methods is high-resolution electron microscopy with negative staining. NanoVan is an intermediate density stain which is used to define the edges of particulate specimens in suspension for electron microscopic observation. It has a highly amorphous structure and fine grain, which provides maximum clarity and least interference in the observation of ultrastructural features at very high resolution. NanoVan is ideal for use with smaller gold labels such as Nanogold® because the stain is less electron-dense than other negative stains such as uranyl acetate or lead citrate, so sufficient contrast is produced between the gold particle, their environment, and the negative stain to differentiate them. Our negative stain reagents are also useful for studies of virus and protein ultrastructure.

    These reagents have several advantages over the commonly used uranyl acetate negative stain:

    • NanoVan and Nano-W are completely miscible: they may be mixed in different proportions to give any desired intermediate stain density.
    • Near-neutral pH results in better ultrastructural preservation.
    • NanoVan is less susceptible to electron beam damage than uranyl acetate.
    • Fine grain allows high imaging resolution.

    [Negative Staining - Principle and Examples (41k)]

    Left: Schematic showing how negative stains work. Right: high-resolution electron micrographs obtained using a scanning transmission electron microscope. (a) Tobacco Mosaic Virus (TMV) negatively stained with 2 % uranyl acetate; (b) TMV stained with 1 % methylamine vanadate (NanoVan); both samples imaged with a dose of 104 eI/nm2. Original full width 128 nm for each image. (c) Side view of groEL (large arrow) labeled with 1.4 nm gold cluster (Nanogold, small arrow) imaged in methylamine vanadate. Note clear visibility of subunit structure and gold cluster. Full width 128 nm. Specimen kindly provided by A. Horwich, Yale University.

    Shim and co-workers recently used NanoVan to support their 2-dimensional infrared spectroscopic and site-specific isotope labeling study to investigate and measure development of secondary structures for 6 residues during the aggregation process of the 37-residue polypeptide associated with type 2 diabetes, the human islet amyloid polypeptide (hIAPP). The researchers used 13C18O labeling of the backbone carbonyl groups to resolve single amide I stretches, using the shifted infrared spectra to monitor the structures and kinetics of individual residues in hIAPP. Six separate samples of hIAPP were prepared, each containing a single 13C18O-labeled residue; from comparison with NMR spectra and changes in the infrared vibrations, changes in the configurations of the peptides could be inferred.

    Fibril morphology was observed by transmission electron microscopy (TEM) for comparison. TEM images were taken of a 3-µL aliquot of fibril solutions generated from 3 peptides that are unlabeled, and Ala-13-, and Leu-27-labeled. Fibril samples were prepared in the same way as those used for the 2D IR spectra, after incubating at least 3 days to complete aggregation: The peptide was dissolved in deuterated hexafluoro-isopropanol (d-HFIP) at a concentration of 0.8 mM. An aliquot of the d-HFIP stock solution was evaporated under a stream of N2 gas to generate a film. Amyloid formation was initiated by redissolving this film in 20mM deuterated potassium phosphate buffer at pH 7, at a final peptide concentration of 1 mM. The aliquot was loaded onto a polyvinyl butyral-coated copper grid, then stained with Nano-W (methylamine tungstate). Excess stain was blotted off, and the sample was allowed to air dry. Images were then acquired using a Philips CM 120 microscope with an accelerating voltage of 80 kV. A common morphology was found of long fibrils with a mean diameter of 10 nm for all 3 fibril samples prepared from 3 differently labeled peptides (unlabeled, labeled at Ala-13, and labeled at Leu-27), similar to previously reported ones prepared by other methods.

    By monitoring the kinetics at 6 different labeled sites, the authors found that the peptides initially develop well-ordered structure in the region of the chain that is close to the ordered loop of the fibrils. This was followed by formation of the 2 parallel beta-sheets with the N-terminal beta-sheet likely forming before the C-terminal sheet. This experimental approach provides a detailed view of the aggregation pathway of hIAPP fibril formation, and also a general approach to the study of other amyloid forming proteins that does not require the use of structure-perturbing labels.

    Reference:

    • Shim, S. H.; Gupta, R.; Ling, Y. L.; Strasfeld, D. B.; Raleigh, D. P., and Zanni, M. T.: Two-dimensional IR spectroscopy and isotope labeling defines the pathway of amyloid formation with residue-specific resolution. Proc. Natl. Acad. Sci. USA, 106, (2009).

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    Nanogold® with Gold Enhancement, STED and CD44 Expression

    Gold enhancement is an alternative to silver enhancement, developed by Nanoprobes. Gold enhancement works similarly to silver enhancement, except that it is gold - instead of silver - that is deposited onto colloidal gold or gold cluster labels. This catalytic enlargement and enhancement process produces enlarged particles for electron microscopic observation, and dark staining for light microscopy, blotting and optical applications.

    Gold enhancement has important advantages over silver enhancement for several applications:

    • Cleaner signals with lower background for light microscopy and blotting.
    • Osmium etch resistance for EM: may safely be used before any strength osmium tetroxide.
    • Compatible with physiological buffers: does not precipitate with halides.
    • Compatible with metal substrates for cell culture or biomaterials.
    • Less pH sensitive than silver enhancement: can be used in a wider pH range.
    • Better for SEM Visualization: much stronger backscatter signal than silver.
    • Near neutral pH for best ultrastructural preservation. Low viscosity for ease of use.

    Gorlewicz and group demonstrated some of these advantages in their recent paper in Neurobiology of Disease, in which they used high resolution morphological methods, including both stimulated emission depletion (STED) superresolution fluorescence microscopy and gold enhanced Nanogold immunoelectron microscopic labeling, tissue fractionation and RT-PCR to localize the expression of the multifunctional cell surface glycoprotein CD44, which regulates cellcell and cellmatrix interactions in a variety of tissues. This protein has been found to be expressed in glial cells of developing peripheral nerves, where it takes part in signaling mediated by ErbB class of receptors for neuregulins, but is no longer present in adult peripheral nerves.

    For immunoelectron microscopy, a pre-embedding method was performed in 20 µm-thick sections of the diaphragm muscle excised from adult Sprague-Dawley rats, fixed in 4% freshly depolymerized paraformaldehyde solution and cryoprotected 2.3 M sucrose solution, cut into small pieces (c.a. 1 mm in width) and snap-frozen in liquid nitrogen. The 12 ?m-thick cryosections were produced using a cryo-chamber attached to the ultramicrotome according to the method of Tokuyasu, and immunolabeled using one of two primary antibodies: either a rabbit polyclonal (Clone plc332) raised against a sequence around amino acid 39 of the human CD44, diluted 1:100, or mouse monoclonal (Clone OX-49) recognizing the N-terminus of the rat CD44, diluted 1:200. Permeabilization was achieved by repeated freezing-thawing rather than the application of detergent; labeling was conducted by sequential incubations in a mixture of primary reagents, one the anti-CD44 antibody, and the other a specific antibody (from a different species) used for double-label fluorescent imaging (overnight at 4°C), followed by Nanogold-labeled secondary antibody, followed in turn by gold intensification using GoldEnhance EM for detection. Correlative fluorescent staining for AChRs had been used to mark the area of interest (neuromuscular junction, or NMJ) before tissue was postfixed in osmium tetroxide, dehydrated and flat-embedded in Spurr epoxy-resin. The marked areas were excised, mounted in an ultramicrotome, and cut into ultrathin sections; these were mounted on copper grids and contrasted with lead citrate and uranyl acetate according to the routine procedure before electron microscope examination. Images of four distinct NMJs from a single animal were photographed at various magnifications. Statistical evaluation of the immunolabeling was performed using Chi-squared test, based on the observed and expected gold grain counts over the given compartments.

    		            

    [Gold enhancement mechanism (60k)]

    Enhancement of Nanogold® by GoldEnhance: mechanism. Final particle size is controlled by enhancement time; particles may be enlarged to sizes between 3 nm (1-2 minutes) and 50 nm or larger (10 minutes and longer).

    Combining microscpic results with tissue fractionation and RT-PCR, showed that CD44 is strongly expressed in terminal Schwann cell (TSC) at the neuromuscular junction (NMJ) of adult rat skeletal muscle. Since CD44 is also expressed by Schwann cells of the non-myelinated Remak bundles of the proximal peripheral nerves, this suggests that it is a marker of non-myelinating Schwann cell subpopulation. Analysis of transgenic rats bearing a mutated superoxide-dismutase gene (SOD1G93A) causing familial amyotrophic lateral sclerosis (ALS) revealed that TSC activation and morphological plasticity at the NMJ, caused by ongoing denervationreinnervation is associated with a strong increase in CD44 expression. Notably, CD44 immunoreactivity is present in fine axon-escheating processes of the glial cells that guide reinnervation. In addition, the authors found that both in normal and SOD1G93A muscle, CD44 expressed in TSC partially colocalizes with immunoreactivities of neuregulin receptors ErbB2 and ErbB3, apparently reflecting a physical interaction evidenced by co-immunoprecipitation and fluorescence resonance energy transfer (FRET) analysis between CD44 and ErbB3. TSC activation upon ALS-like neurodegeneration results in significant increase in molecular proximity of CD44 and ErbB3: this may have an impact on glial plasticity at the NMJ.

    Reference:

    • Gorlewicz, A.; Wlodarczyk, J.; Wilczek, E.; Gawlak, M.; Cabaj, A.; Majczynski, H.; Nestorowicz, K.; Herbik, M. A.; Grieb P.; Slawinska, U.; Kaczmarek, L., and Wilczynski, G. M.: CD44 is expressed in non-myelinating Schwann cells of the adult rat, and may play a role in neurodegeneration-induced glial plasticity at the neuromuscular junction. Neurobiol. Dis., 34, 245-58 (2009).

    An example of electron microscopic immunolableing using Nanogold-Fab' enhanced with GoldEnhance EM is shown below, together with a light microscopic image showing the comparison with organic enzyme chromogens.

    [GoldEnhance: TEM labeling and ISH Visualization vs. DAB (126k)]

    (a): Pre-embedding immunolabeling using Nanogold-Fab and GoldEnhance EM, showing uniform enlarged particles (image courtesy of A. A. Mironov). (b): DAB vs. GoldEnhance-Nanogold for in situ hybridization: Formalin-fixed serial paraffin sections of cervical carcinoma, in situ hybridized for HPV-16/18 (contains one to a few copies) using a biotinylated probe (bar = 10?m). (1) DAB-peroxidase; (2) Nanogold-streptavidin with GoldEnhance (courtesy of G. W. Hacker, Medical Research Coordination Center, University of Salzburg).

    GoldEnhanceTM is also better than silver enhancement for some light microscopy applications. In in situ hybridization, GoldEnhanceTM LM produces lower background that silver enhancement, and a cleaner, darker signal. This provided an early example of Chromogenic in situ hybridization (CISH), which is fast becoming an accepted alternative to fluorescent in situ hybridization (FISH). Gold Enhancement is only one of a number of detection technologies developed by Nanoprobes that provide higher sensitivity, lower copy number detection and clearer signals than enzyme chromogens such as DAB; others include Nanogold® with silver acetate autometallography and Enzyme metallography (EnzMetTM).

    Reference:

    • Tubbs, R.; Pettay, J.; Skacel, M.; Powell, R.; Stoler, M.; Roche, P., and Hainfeld, J.: Gold-Facilitated in Situ Hybridization: A Bright-Field Autometallographic Alternative to Fluorescence in Situ Hybridization for Detection of HER-2/neu Gene Amplification. Am. J. Pathol., 160, 1589-1595 (2002).

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    Guidelines for Custom Syntheses and Contract Research

    The explosion of nanotechnology applications has created a large demand both for novel gold nanoparticle-based reagents and conjugates. As one of the first companies in this field, Nanoprobes owns a variety of proprietary core technologies that give us the unique capability to address these applications, so if you are looking for a new type of nanoparticle or probe, please check us out.

    To make it easier to assess the feasibility of custom syntheses and provide quotations, we have updated our guidelines.

    General Information

    Gold cluster and nanoparticle reagents developed by Nanoprobes, Incorporated are attached to biological molecules by covalent cross-linking, and may be used to label many molecules with suitable reactive functionalities. Our gold labels have been conjugated to proteins, lectins, oligoncleotides, peptides, lipids, biotin and cytoskeletally active probes such as modified phalloidins. If you require a gold conjugate or other molecule that is not in our catalog, we may consider preparing it as a custom synthesis or contract research project.

    Our capabilities include:

    • Preparation of chemically reactive gold nanoparticles.
    • Bioconjugation of gold nanoparticles and purification of nanoparticles and conjugates.
    • Characterization, blot and light microscopy testing of conjugates.

    Types of custom work and payment arrangements

    Custom Synthesis, in which we contract to deliver a specified amount of product, is generally offered for preparations that are substantially similar to products in our catalog. These include the following reactions:

    • Nanogold® labeling of polyclonal or monoclonal antibodies.
    • Nanogold labeling of proteins.
    • Preparation of Nanogold with multiple reactive groups.
    • Preparation of Nanogold in alternative buffers or solvents.

    We will also be pleased to provide quotations for larger quantities or standing orders for any of our products.

    Contract research, in which we contract to undertake research and require payment whether or not it produces the desired outcome, is generally necessary for conducting substantially novel procedures or preparing new entities. This might include synthesis of gold nanoparticles in alternate sizes, or with novel functionality, preparation of goldlabeled oligonucleotides, peptides, lipids or small molecules, or novel fluorescent and gold-labeled conjugates.

    While we will be glad to consider such requests, our time and resources available to undertake such projects are limited. It should be noted that new syntheses frequently require more work than anticipated.

    Successful Custom Syntheses

    Examples of custom products that have been described in the scientific literature include:

    • Tetrairidium labeling reagent:
      A very small cluster, containing four iridium atoms, has been prepared as a maleimide and used to derivatize virus subunits for localization during cryo-EM and image reconstruction, and as a label for infrared microspectroscopy.

    • Nanogold® and Texas Red-labeled dextran
      A 10,000 MW amino-functionalized Texas Red-labeled dextran was conjugated with Nanogold and used as a neuronal tracer.

    Guidelines

    Before requesting a custom synthesis quotation, we recommend that you consider our Nanogold or undecagold labeling reagents, which you may use to label a wide variety of molecules. See p. xx for more details. We are glad to advise on the preparation of gold conjugates with other molecules that are not described in this catalog: you can contact us by telephone at 1-877-447-6266 (US & Canada) or ++(01-631) 205-9490 (others), or by e-mail at tech¤nanoprobes.com.

    Please note that our researchers may not be familiar with your probe or application. We can usually respond more quickly if you tell us about the molecule you wish to label:

    • Molecular weight: labeling reactions use specific ratios of gold to probe. Molecular weight is used to calculate how much reagent to use, and how to separate the product.

    • Optical density or UV/visible absorbtion: Extinction coefficients are known for Nanogold and undecagold. They are used to monitor the reaction and to calculate the labeling efficiency.

    • References for labeling with other tags: Successful labeling experiments or protocols using enzymes or fluorophores are often a good guide to what will work with our probes and help us avoid potential problems.

    • Molecular structure and functional groups: gold labeling is usually more successful when the gold is attached at a unique site located away from the binding region, so that it does not perturb the activity of your probe. We use the structure to identify the best sites and reagents for labeling.

    • Solubility: The solubility of some conjugates can differ significantly from that of either the conjugate biomolecule or the gold cluster. Information on solubility and tolerance for organic solvents such as DMSO or isopropanol can help avoid or minimize reagent loss and solubility testing.

    Terms and Conditions

    Custom syntheses and contract research projects and products prepared by such projects are non-returnable and non-refundable. Products are intended for laboratory research purposes and may not be used for other purposes, including but not limited to human clinical trials or commercial purposes, specifically the resale of our Products to unaffiliated third parties without written approval from Nanoprobes.

    Nanoprobes reserves all of its rights under any patents or other intellectual property rights covering the use, modification, or combination with other materials of its Products, technology or know-how. No rights, title or interest in any new products or materials developed during a custom synthesis or contract research project are granted or implied to the buyer by purchasing a product prepared as a custom synthesis, or entering into a contract for a research project.

    Larger or more speculative projects in which significant patented or proprietary material, knowledge, expertise or know-how is supplied by both parties may require a separate agreement specifying disposition of rights to any discoveries, and discussion of such projects may require entering into a confidentiality agreement beforehand.

    Nanoprobes has not tested any products for safety and efficacy in foods, drugs, biologics, medical devices, in vitro diagnostics, cosmetics or other clinical or human uses, unless expressly stated in our literature furnished with products.

    More Information

    For more information or to discuss a specific project, contact us by telephone at 1-877-447-6266 (US & Canada) or ++(01-631) 205-9490 (others), or by e-mail at tech@nanoprobes.com. Request a quotation online with our custom synthesis request form.

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    Other Recent Publications

    More pre-embedding Nanogold labeling with HQ Silver enhancement was described by Bauer, Lujan and group in their investigation into neuropathic pain, reported in the Journal of Neuroscience. Neuropathic pain results from damage to the peripheral sensory nervous system. In several animal models, the calcium channel subunit alpha2delta-1 is upregulated in dorsal root ganglion (DRG) neurons, and this is causally related to the onset of allodynia, in which a non-noxious stimulus becomes painful. The therapeutic drugs gabapentin and pregabalin (PGB), which are both alpha2delta ligands, have antiallodynic effects, but their mechanism of action has not been fully determined. The authors used an in vivo rat model of neuropathy, unilateral lumbar spinal nerve ligation (SNL), to investigate this by characterizing the distribution of alpha2delta-1 in DRG neurons at the light and electron microscopic level. For electron microscopic observation, a preembedding immunogold method was used: animals were perfused with 4% paraformaldehyde, 0.05% glutaraldehyde, and 15% (v/v) saturated picric acid in 0.1 M phosphate buffer. Tissue was postfixed overnight, and coronal slices of 60 µm thickness cut on a vibratome. Free-floating sections were blocked in Tris-buffered saline (TBS) with 10% goat serum (GS) for 1 hour at room temperature, incubated in TBS with 1% GS and anti-alpha2delta-1 antibody at 1:100 for 48 hours, then washed with TBS and incubated in TBS with 1% GS and 1 : 100 dilution Nanogold$#174; goat anti-mouse IgG for 3 hours. After washing with phosphate-buffered saline (PBS), sections were postfixed with 1% glutaraldehyde in PBS for 10 minutes, washed in double-distilled water, and enhanced with HQ Silver. Sections were then treated with osmium tetraoxide (1% in 0.1 M PB), block-stained with uranyl acetate, dehydrated through a graded series of ethanol, and flat-embedded on glass slides in Durcupan resin. Regions of interest were cut close to the surface of each block at 7090 nm using an ultramicrotome, collected on 200-mesh nickel grids, and stained with 1% aqueous uranyl acetate followed by Reynoldss lead citrate. For controls, primary antibodies were either omitted or replaced with 5% (v/v) normal mouse serum: under these conditions, no selective labeling was observed. In addition, some sections were incubated with gold-labeled secondary antibodies without silver intensification, which resulted in no metal particles in the sections.

    On the side of the ligation, alpha2delta-1 was found to be increased in the endoplasmic reticulum of DRG somata, in intracellular vesicular structures within their axons, and in the plasma membrane of their presynaptic terminals in superficial layers of the dorsal horn. Chronic PGB treatment of SNL animals, at a dose that alleviated allodynia, markedly reduced the elevation of alpha2delta-1 in the spinal cord and ascending axon tracts. In contrast, it had no effect on the upregulation of alpha2delta-1 mRNA and protein in DRGs. In vitro, PGB reduced plasma membrane expression of alpha2delta-1 without affecting endocytosis. The authors concluded that the antiallodynic effect of PGB in vivo is associated with impaired anterograde trafficking of alpha2delta-1, resulting in its decrease in presynaptic terminals: this reduces neurotransmitter release and spinal sensitization, an important factor in the maintenance of neuropathic pain.

    Reference:

    • Bauer, C. S.; Nieto-Rostro, M.; Rahman, W.; Tran-Van-Minh, A.; Ferron, L.; Douglas, L.; Kadurin, I.; Sri Ranjan, Y.; Fernandez-Alacid, L.; Millar, N. S.; Dickenson, A. H.; Lujan, R., and Dolphin, A. C.: The increased trafficking of the calcium channel subunit alpha2delta-1 to presynaptic terminals in neuropathic pain is inhibited by the alpha2delta ligand pregabalin. J. Neurosci., 29, 4076-4088 (2009).

    Meanwhile, Shiuh-Bin Fang and co-workers, reporting in the , describe an easy gold-based screening test that identifies Salmonella in 2 hours after colony-print: the test procedure is based on the transfer of surface cells of the colonies to a nitrocellulose membrane, followed by visualization of the transferred cells with gold nanoparticle - anti-Salmonella antibody conjugates to facilitate the selectivity. On Hektoen agar, 134 stool samples containing black or crystalloid colonies were identified using the new assay. The test is read by eye without any equipment, such as a microscope: red dots corresponding to Salmonella are observed. After colony-print test, 22 of the isolates were correctly identified as Salmonella to achieve 100% sensitivity. 111 samples were correctly identified as non-Salmonella, but one was incorrectly identified as Salmonella, giving a specificity of 99.1%. The method is simple, straightforward, inexpensive, and fast: it can be easily applied to the routine workload of clinical laboratories, and may be very useful when large numbers of fecal samples require rapid screening and diagnosis.

    Reference:

    • Fang, S. B.; Tseng, W. Y.; Lee, H. C.; Tsai, C. K.; Huang, J. T., and Hou, S. Y.: Identification of Salmonella using colony-print and detection with antibody-coated gold nanoparticles. J. Microbiol. Methods., 77, 225-228 (2009).

    Reminding us that gold nanoparticles also work well in electrochemical systems, Vig and colleagues report an impedimetric immunosensor based on colloidal gold and silver electrodeposition for the detection of aflatoxin M1 (AFM1) in their recent paper in Sensors and Actuators B: Chemical. An procedure similar to indirect competitive ELISA procedure was performed on screen-printed electrodes (SPEs) in the presence of anti-AFM1 gold-labeled antibodies. Silver was chronoamperometrically electrodeposited at a fixed applied potential for a determined period of time to amplify the signal. The calculated charge transfer resistance (Rct) was found to correlate well with the concentration of AFM1. The linear working range of the described AFM1 immunosensor ranged between 15 and 1000 ng/L with a limit of detection (LoD) equal to 15 ng/L (R.S.D. = 20%). Impedimetric results were confronted with linear sweep voltametry (LSV) and corresponded well to this technique.

    Reference:

    • Viga, A.; Radoib, A.; Muñoz-Berbel,X.; Gyemantc, G., and Marty, J.-L.: Impedimetric aflatoxin M1 immunosensor based on colloidal gold and silver electrodeposition Sens. Act. B Chem., B138, 214-220(2009).

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