N A N O P R O B E S     E - N E W S

Vol. 9, No. 9          September 30, 2008

This probe is particularly important for high-resolution labeling of components within small systems, such as the identification of protein subunits in complexes and organelles. It has significant advantages over conventional antibody probes:

• Higher labeling resolution. The nitrilotriacetic acid - Ni(II) chelate is much smaller than an antibody or protein, and therefore when it is bound, the gold is much closer to its target. This makes NTA-Ni(II)-Nanogold ideal for localizing sites in protein complexes or other macromolecular assemblies at molecular resolution.

• Better penetration: because it is so small, NTA-Ni(II)-Nanogold can more easily penetrate into specimens and access sterically restricted sites within specimens, and perturbs their ultrastructure less. In some systems it may be used with stronger fixation or less permeabilization, enabling labeling with better ultrastructural preservation.

• NTA-Ni(II)-Nanogold is prepared using a modified gold particle, with very high solubility and stability. At 1.8 nm in size, it is readily visualized by electron microscopy.

• Binding constants for Ni(II)-NTA are very high due to the combination of the chelate effect of multiple histidine binding, and target binding of multiple Ni(II)-NTA functionalization. Dissociation constants are estimated to be between 10^-7 to 10^-13 M^-1. For many applications, this provides binding strengths comparable to antibodies.

Applications include:

• High-resolution labeling of proteins, protein complexes or organelles containing recombinant His-tagged proteins for EM1 or STEM2 localization.
• Labeling and molecular localization of two different subunits of Photosystem II.
• "Universal" pre-embedding labeling of His-tagged proteins in tissue sections for electron microscopic observation.
• identifying His-tagged proteins in fractions during Ni-NTA-column purifications.
• Detection of recombinant His-tagged proteins on blots and in gels.
• Heavy atom labeling of regular structures for image analysis and structure solution.

Young and co-workers provided an excellent demonstration of the advantages of this higher resolution in their recent paper in Journal of Biological Chemistry, in which they used NTA-Ni(II)-Nanogold labeling at the electron microscopic level in conjunction with fluorescence resonance energy transfer (FRET) at the light level to determine the molecular shape, architecture, and size of P2X₄ receptors. Seven types of P2X receptor subtypes are found in mammals; they display a diverse tissue distribution, and play key roles in physiological processes that include neurotransmission, sensory transduction, inflammation, and cardiovascular regulation. They are mostly localized on the plasma membrane, but in some species (such as Dictyostelium) they may also carry out physiological functions within intracellular membranes. They are the third major superfamily of ligand-gated ion channels, but little is known about their 3-dimensional structure.

The authors studied the structure of P2X₄ receptors purified from HEK293 or HEK293T cells transiently expressing the appropriate wild-type and fluorescently labeled P2X receptor subunits. The efficiency of fluorescence resonance energy transfer (FRET) between subunits within homomeric P2X receptors bearing yellow (YFP) or cyan (CFP) fluorescent protein tags at their C termini was measured; FRET efficiency measurements indicated that the distance between the C-terminal tails of P2X₄ receptors was 5.6 nm. They then used electron microscopy with NTA-Ni(II)-Nanogold labeling to determine the orientation of the P2X4 relative to the associated membrane. Purified P2X₄-His₁₀ in 0.1% phosphate-buffered saline containing 1% (w/v) beta-D-dodecyl maltoside (DDM) was incubated for 10 minutes at room temperature with a 10 : 1 molar excess of NTA-Ni(II)-Nanogold. Samples were then adsorbed onto grids and washed with NTA-Ni(II)-Nanogold solution to prevent unbinding of the protein-associated gold particles. After transmission electron microscopy and image processing, 1966 gold-labeled P2X₄-His₁₀ particles (in 48 x 48 pixel boxes) and 2862 lectin-labeled particles (in 64 x 64 pixel boxes) were manually selected. Three-dimensional structures were generated using the refined P2X₄ structure as a starting model, followed by eight rounds of iterative refinement.

Single particle analysis of purified P2X4 receptors was used to determine the three-dimensional structure at a resolution of 21Å; the results indicated C3 symmetry, and showed human P2X4 to be a globular torpedo-like molecule with an approximate volume of 270nm³ and a compact, propeller-shaped ectodomain. The orientation of the particle with respect to the membrane was assigned by labeling the intracellular C termini with 1.8-nm gold particles. In the labeled structure, three strong negative densities corresponding to the 1.8-nm diameter gold spheres were clearly averaged into the density map toward the narrow end of the molecule: this confirmed that the propeller-like structure at the top of the molecule corresponded to the ectodomain. The distance between the centers of the gold particles was 6.1 nm, which agreed well with the distance of 5.6 nm obtained from FRET experiments. To confirm the orientation, the particle was also labeled with L. culinaris lectin. The lectin-labeled particle clearly displayed two regions of additional protein density per monomer at the propeller end of the molecule. These data provide the first views of the architecture, shape, and size of single P2X receptors, and significantly increase our understanding of an important family of ligand-gated ion channels.

Reference:

• Young, M. T.; Fisher, J. A.; Fountain, S. J.; Ford, R. C.; North, R. A., and Khakh, B. S.: Molecular Shape, Architecture, and Size of P2X4 Receptors Determined Using Fluorescence Resonance Energy Transfer and Electron Microscopy. J. Biol. Chem., 283, 26241-26251 (2008).
  • Abstract (courtesy of the Journal of Biological Chemistry).
  • reprint (PDF) (courtesy of the Journal of Biological Chemistry).

More information:

• Ni-NTA-Nanogold: Catalog information
• Product information, instructions and protocols
• Ni-NTA-Nanogold: References
• Ni-NTA-Nanogold: Technical help
• Our paper from Microscopy and Microanalysis 2002

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Nanogold® for Labeling Synthetic and Natural Oligonucleotides

Labeling of synthetic oligonucleotides

The most straightforward oligonucleotides to label are synthetic, because you can introduce modifications during synthesis to direct labeling. You can obtain the DNA you wish to label with a suitable reactive group for labeling, such as a thiol or an aliphatic primary amine or a thiol, incorporated using specially modified phosphoramidites such as those supplied by Glen Research; this company supplies a guide to modification and labeling, and most oligonucleotide suppliers will offer this type of modification as an option. Find out from your supplier which of these groups is best inserted at the position you wish to label with Nanogold. you can then label the finished oligonucleotide using Monomaleimido Nanogold to label thiol sites, or Mono-Sulfo-NHS-Nanogold to label amino- sites. Some examples of this approach are given below:

Once you have the modified oligonucleotide, you can label with Nanogold in your laboratory. Thiol-modified oligonucleotides may require deprotection or reduction to activate the thiol group; the reducing agent (dithiothreitol, DTT, or mercaptoethylamine hydrochloride, MEA) must be separated from the reduced oligonucleotide by gel filtration prior to use, otherwise it will react with the Monomaleimido Nanogold and prevent labeling. The oligonucleotide is then mixed with reconstituted Monomaleimido Nanogold at a pH between 6.0 and 7.0, incubated overnight at 4°C, then separated next day by gel filtration or an alternative chromatographic method, or by gel electrophoresis.

References for labeling oligonucleotides with Monomaleimido Nanogold:

• Alivisatos, A. P., Johnsson, K. P., Peng, X., Wilson, T. E., Loweth, C. J., Bruchez, M. P., Jr., and Schultz, P. G.: Organization of 'Nanocrystal Molecules' using DNA. Nature, 382, 609-611 (1996).
  • Abstract (courtesy of PubMed (Medline)).

• Dubertret, B., Calame, M., and Libchaber, A.; Nat. Biotechnol., 19, 365-370 (2001).
  • Abstract (courtesy of PubMed (Medline)).

Reference for labeling oligonucleotides with Mono-Sulfo-NHS- Nanogold:

• Hamad-Schifferli, K.; Schwartz, J. J.; Santos, A. T.; Zhang, S., and Jacobson, J. M.: Remote electronic control of DNA hybridization through inductive coupling to an attached metal nanocrystal antenna. Nature, 415, 152-155 (2002).
  • Abstract (courtesy of PubMed (Medline)).

Modifiers are also available that can be used to introduce carboxyls or aldehydes; these may be labeled with Monoamino Nanogold once synthesis is complete.

Other approaches are feasible, and these are discussed in a section on oligonucleotide labeling in the technical help section for Nanogold labeling reagents on our web site. In addition, our online Guide to Gold Cluster Labeling explains how to optimize labeling and separation procedures for different types of conjugate biomolecules and different applications, and the options for oligonucleotide labeling have been discussed in detail in a previous issue of our newsletter. Reactions include:

• 5'-Labeling with Monoamino Nanogold (for all oligonucleotides).
• Thiolation of 5' terminus and Labeling with Monomaleimido-Nanogold (for all oligonucleotides).
• Include a hapten such as biotin in your oligonucleotide, then label with Nanogold-Streptavidin

If you need help in finding a suitable synthesis, we would be glad to advise; please contact our technical support with your questions.

Labeling of plasmids and other naturally occuring oligonucleotides

If you are labeling a synthetic oligonucleotide, functional groups for selective labeling are readily introduced during oligonucleotide synthesis using modified phosphoramidites. What happens, though, if you are working with a plasmid or other enzymatically-generated or naturally occurring oligonucleotide, and you can't use modified phosphoramidites? You have several options, including the following approaches:

• Use a modified nucleotide during plasmid preparation. A variety of modified bases are available that can be incorporated by polymerases or other enzymes. In a previous article, we reported how Willner and group used a 10 : 1 mixture of unmodified dUTP with an amino-modified form of dUTP, 5-[3-Aminoallyl]-2'-deoxyuridine 5'-triphosphate (amino-dUTP) to prepare amino-modified DNA in cancer cells. This provides a primary aliphatic amine, which may be labeled using Mono-Sulfo-NHS-Nanogold. Malecki used nick translation with a biotin-conjugated dUTP to incorporate biotin; the biotinylated DNA was then incubated with Nanogold-streptavidin as part of the transfection complex. Boublik and co-workers used 2-thiocytidine. If you incorporate a small amount of one of these into your plasmid preparation mixture, it will be incorporated and the reactive groups will be introduced. Trilink Biotechnologies offer a wide range of modified and functionalized nucleotides that may be suitable for this type of modification. The strategy is shown in Scheme 1:

  

  Incorporation of 5-[3-Aminoallyl]-2'-deoxyuridine 5'-triphosphate (amino-dUTP) into plasmid, followed by labeling with Mono-Sulfo-NHS-Nanogold at an incorporated amine site.

  If present in small proportions (for example, if about 20% of the nucleotide is in the modified form) will be incorporated with relatively low frequency into the plasmid, facilitating lower-density labeling which has less likelihood of affecting transfection. The actual proportion of modified nucleotide that you use will be determined by the size of the construct you are preparing and the number of labeling sites you wish to introduce.

• Use a photoreactive cross-linker to introduce a reactive site or a hapten into the completed plasmid. A good source of cross-linking reagents with a wide variety of functionalities is Pierce, who provide a wide range of different types of reactivity, functionality and cleavability. Pierce also provides an online cross-linker selection guide that you can use to identify the best cross-linkers for your application. This tool lets you specify the reactivities and functionalities to be introduced; select "non-selective/photoreactive" for "Functional Group Reactive Toward 1" and "Amine" for "Functional Group Reactive Toward 2;" this will provide you with a list of cross-linkers that you can use to introduce amines. If you then also select "Cleavable by thiols" under "cleavability," the reagents you select will include a disulfide within the chain, or an alternative group which, when cleaved, provides a thiol suitable for labeling with Monomaleimido Nanogold.

  Another example of this approach is the Fast-tag system from Vector Laboratories; this provides another method for introducing thiols which can then be labeled with Monomaleimido Nanogold. An alternative supplier of novel cross-linking reagents is Molecular Biosciences; this company also sorts its products by reactivity.

  You can also use this approach to biotinylate the oligonucleotide, using a photoreactive biotinylation reagent. The biotinylated plasmid is then localized with Nanogold-streptavidin. This reagent provides high sensitivity and specificity for in situ hybridization; it is described in full, with references, links and full protocols, in a special report on our web site.

• Use Positively Charged Nanogold, which binds to the negatively charged oligonucleotide backbone, to selectively decorate the plasmid. This approach has been used to decorate DNA for STEM visualization, and when used with linear DNA, provides a potential method for preparing DNA-based conductive nanowires. In our paper from Microscopy and Microanalysis 2001, we have described the preparation of labeled DNA by incubating the DNA with a solution of Positively Charged Nanogold. The reaction is straightforward: positively charged Nanogold may be mixed with double-stranded DNA either with the DNA immobilized on a grid, or in solution, and it will bind through the interaction of the positive charges on the Nanogold with the negative charges on the DNA backbone. A near neutral to slightly acidic pH (6 to 7.5) may work best. Unbound Nanogold may be removed either by washing the labeled DNA when it is bound to a support or a surface, or by chromatographically separating the labeled DNA using gel filtration over a column such as Amersham Pharmacia Superose-6 or Superose-12. Some trial-and-error adjustment of the ratio of Nanogold to DNA may be necessary if the reaction is carried out in solution: some precipitation may be observed if the Nanogold binds to more than one DNA molecule, but this may be eliminated by increasing the excess of Nanogold.

References:

• Functionalization of plasmids:

  Malecki, M.: Preparation of plasmid DNA in transfection complexes for fluorescence and spectroscopic imaging. Scanning Microsc. Suppl. (Proc. 14th Pfefferkorn Conf.); Malecki, M., and Roomans, G. M. (Eds.). Scanning Microscopy International, Chicago, IL, 10, 1-16 (1996).

• Functionalization and undecagold labeling of tRNA:

  Hainfeld, J. F.; Sprinzl, M.; Mandiyan, V.; Tumminia, S. J., and Boublik, M.: Localization of a specific nucleotide in yeast tRNA by scanning transmission electron microscopy using an undecagold cluster. J. Struct. Biol., 107, 1-5 (1991).

  • Abstract (courtesy of Science Direct).

RNA Labeling

RNA differs from DNA in that it contains a cis-1,2-dihydroxy sugar moiety, which is readily oxidized by may be conducted by using periodate to oxidize the 1,2-dihydroxy group in the sugar moiety. This is readily oxidized to a dialdehyde, and this in turn reacts readily with Monoamino Nanogold. This reaction is described in an application note on our web site; it was also used to label ATP.

Reference:

• Hainfeld, J. F.; Liu, W., and Barcena, M.: Gold-ATP. J. Struct. Biol., 127, 120-134 (1999).
  • Abstract (courtesy of Science Direct).

If this reaction is not appropriate to your situation, you can incorporate a reactive group enzymatically. Blechschmidt and co-workers used tRNA(Phe) from Escherichia coli, enzymatically aminoacylated with phenylalanine in the reaction catalyzed by phenylalanyl-tRNA synthetase, for labeling with undecagold. In a similar reaction, Boublik and co-workers selective labeled a sulfhydryl group on 2-thiocytidine, enzymatically inserted using t-nucleotidyl transferase at position 75 at the 3' end of yeast tRNA(Phe), with Monomaleimido Undecagold.

References:

• Blechschmidt, B., Shirokov, V., and Sprinzl M.: Undecagold cluster modified tRNA (Phe) from Escherichia coli and its activity in the protein elongation cycle. Eur. J. Biochem., 219, 65-71 (1994).
  • Abstract (courtesy of Blackwell-Synergy).
  • Reprint (PDF) (courtesy of Blackwell-Synergy).

• Hainfeld, J.F., Sprinzl, M., Mandiyan, V., Tumminia, S.J. and Boublik, M. Localization of a specific nucleotide in yeast tRNA by scanning transmission electron microscopy using an undecagold cluster. J. Struct. Biol., 107, 1-5, (1991).
  • Abstract (courtesy of Science Direct).

Procedure for enzymatic insertion of 2-thiocytidine using t-nucleotidyl transferase:

• Sprinzl M.; Scheit K.-H., and Cramer, F.: Preparation in vitro of a 2-thiocytidine-containing yeast tRNA Phe -A 73 -C 74 -S 2 C 75 -A 76 and its interaction with p-hydroxymercuribenzoate. Eur. J. Biochem., 34, 306-310 (1973).
  • Abstract (courtesy of Blackwell-Synergy).
  • Reprint (PDF) (courtesy of Blackwell-Synergy).

Medalia and co-workers have described another potentially useful approach: they used ribonucleoside triphosphate analogs with a terminal thiol group attached to the heterocyclic ring for the in vitro transcription of RNAs carrying free thiol groups; these were then labeled with Monomaleimido Nanogold.

Reference:

• Medalia, O.; Heim, M.; Guckenberger, R.; Sperling, R., and Sperling, J.: Gold-Tagged RNA - A Probe for Macromolecular Assemblies. J. Struct. Biol., 127, 113-119 (1999).
  • Abstract (courtesy of Science Direct).

More information:

• Tech help: Nanogold labeling of oligonucleotides
• Catalog information: Nanogold labeling reagents
• Product instructions: Monomaleimido Nanogold
• Product instructions: Mono-Sulfo-NHS-Nanogold
• Product instructions: Monoamino Nanogold
• References: Nanogold labeling of DNA and RNA
• Guide to Gold Cluster Labeling

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

FluoroNanogold and GoldEnhance for Neuroscience

FluoroNanogold fluorescent and gold probes have been used for a number of different correlative microscopy applications:

• Combined and Correlative fluorescence and transmission electron microscopic (TEM) labeling.
• Correlative fluorescence and scanning electron microscopy (SEM).
• Correlative optical and X-ray fluorescence microscopy.

The illustration below shows the structure of Alexa Fluor®* 488 FluoroNanogold and examples of the fluorescent labeling obtained with these probes, as well as combined fluorescent and SEM labeling.

More mundane, but equally significant in terms of time saved, is the use of FluoroNanogold to check that labeling has worked before processing for electron microscopy. In their recent paper in the Proceedings of the National Academy of Sciences of the USA, Patrizi and group used FluoroNanogold for pre-embedding immunolabeling to localize the postsynaptic adhesion molecule neuroligin-2 (NL2) in rat brain sections. GABAergic synapses are crucial for brain function, but the mechanisms underlying inhibitory synaptogenesis are unclear. This study had two goals: to assess in vivo whether the localization of NL2 at GABAergic synapses depends on GABA_A receptors and on the integrity of the postsynaptic apparatus, a major component of which is the scaffolding molecule gephyrin; and to understand to what extent synapse formation requires GABA-mediated neurotransmission. In order to investigate these issues, the authors used cerebellar Purkinje cells (PCs) of GABA_A receptor alpha1 subunit (GABA_Aalpha1) KO mice, which have been shown to lack postsynaptic GABAergic activity: in PCs of GABA_Aalpha1 KO mice, perisomatic synapses are present in normal numbers, whereas dendritic synapses are strongly reduced, and GABAergic axons make mismatched synapses with dendritic spines.

For pre-embedding immunoelectron microscopy, three adult (P30) mice of both genotypes were perfused with 4% paraformaldehyde in phosphate buffer. The cerebella were post-fixed for 2 hours and cut into 70-µm parasagittal sections with a vibratome. The sections were cryoprotected in 30% sucrose, frozen and thawed to enhance antibody penetration, then incubated with anti-NL2 (1 : 2,000 dilution) for 3 days at 4°C. Next, the sections were incubated overnight in FluoroNanogold-Fab conjugates (diluted 1 : 100), then post-fixed in 1% glutaraldehyde (10 minutes), and then gold enhanced as described in the product instructions. Finally, the sections were postfixed with 0.5% osmium tetroxide (15 minutes), block-stained for 25 minutes with 1% uranyl acetate, and embedded in Epon 812. Ultrathin sections were observed and photographed with a JEM-1010 electron microscope (Jeol) equipped with a sidemounted CCD camera.

Postnatal Purkinje cells (PCs) of GABA_Aalpha1 knockout (KO) mice transiently express the alpha3 subunit, leading to the assembly of functional GABA_A receptors and initial normal formation of inhibitory synapses, which are retained until adulthood. However, subsequently, down-regulation of the alpha3 subunit causes a complete loss of GABAergic postsynaptic currents, resulting in a decreased rate of inhibitory synaptogenesis and formation of mismatched synapses between GABAergic axons and PC spines. The electron microscopic results show that the postsynaptic adhesion molecule neuroligin-2 (NL2) is correctly targeted to inhibitory synapses lacking GABA_A receptors and the scaffold molecule gephyrin, but is absent from mismatched synapses, despite innervation by GABAergic axons. This implies that GABA_A receptors are dispensable for synapse formation and maintenance and for targeting NL2 to inhibitory synapses. However, GABAergic signaling appears to be crucial for activity-dependent regulation of synapse density during neuronal maturation.

Reference:

• Patrizi, A.; Scelfo, B.; Viltono, L.; Briatore, F.; Fukaya, M.; Watanabe, M.; Strata, P.; Varoqueaux, F.; Brose, N.; Fritschy, J. M., and Sassoè-Pognetto, M.: Synapse formation and clustering of neuroligin-2 in the absence of GABAA receptors. Proc. Natl. Acad. Sci. USA, 105, 13151-13156 (2008).

• Abstract (courtesy of the Proceedings of the National Academy of Sciences of the USA).

More information:

• Catalog information: FluoroNanogold
• Alexa Fluor® 488 FluoroNanogold-Fab': Product information and instructions
• Alexa Fluor® 488 FluoroNanogold-Streptavidin: Product information and instructions
• FluoroNanogold: Technical help
• FluoroNanogold: References, sorted by application

*® Alexa Fluor is a trademark of Molecular Probes / Invitrogen.

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Post-Embedding Immunoelectron Microscopic Labeling with Nanogold®

• High penetration into cells and tissues.
• High labeling density.
• Close to quantitative labeling of antigenic sites.
• High labeling resolution.

These advantages were recently demonstrated by Hwang and colleagues, who used it to help identify and localize a novel protein, nematocilin, in the mechanosensory cilium of Hydra nematocytes; the study is described in a recent paper in Molecular Biology and Evolution. The cnidocil at the apical end of Hydra nematocytes is a mechanosensory cilium, which acts as a "trigger" for discharge of the nematocyst capsule. The cnidocil, which protrudes from the center of the cnidocil apparatus, is composed of singlet and doublet microtubules surrounding an electron-dense central filament.

Following their identification of 51 nematocyte-specific genes by cDNA microarray and in situ hybridization, the authors investigated the proteins coded by two of these genes (hmp_08523 and hm_04087) which are strongly expressed in the tentacles after the differentiation of the nematocyst capsule is completed. These cDNAs were shown to be derived from 2 paralogous genes encoding lamin-like proteins of 47 kDa, named nematocilin A and B. The authors used immunofluorescence and post-embedding immunoelectron microscopy to localize these proteins and deduce their role.

For post-embedding immunoelectron microscopy, Hydra magnipapillata tentacles were fixed in 0.1 M cacodylate buffer (pH 7.0) with 4% paraformaldehyde and 0.05% glutaraldehyde overnight at 4 °C, washed (3 x 10 minutes) in 0.1 M cacodylate buffer (pH 7.0), dehydrated through an increasing series of ethanol solutions, and infiltrated with medium grade LR White resin; polymerization was achieved by an accelerator compound at 4°C for two days. Ultrathin sections (ca. 70 nm, prepared using an ultramicrotome) were collected on a formvar-coated nickel grid, then treated with a saturated aqueous sodium metaperiodate for 10 minutes, rinsed with distilled water and finally with 0.1 M HCl for 10 minutes. Nonspecific binding was blocked with 1% BSA in 0.1 M cacodylate buffer, pH 7.0, with 0.1% Triton X-100 for 30 minutes. Sections were then exposed to small drops of polyclonal anti-nematocilin antibody (1:100) diluted in blocking solution for 2 hours, washed with 0.1 M cacodylate buffer (pH 7.0), and incubated with a 1 : 100 dilution of Nanogold - goat anti-rabbit IgG for 2 hours. The sections were then washed several times with 0.1M cacodylate buffer (pH 7.0), and the gold particles enhanced using HQ Silver. Finally, the sections were stained with 2% aqueous uranyl acetate followed by 0.4% lead citrate for 10 minutes each and viewed with a transmission electron microscope (TEM) operating at 80 kV.

Negative staining, immunogold labeling and electron microscopy were also carried out for isolated cnidocils. Cnidocils were isolated from 30 to 40 animals by incubation in 7% ethylene glycol in cold M medium for 20 seconds, and collected by centrifugation at 13,000 rpm for 10 minutes. The pellet was resuspended in water before adsorption to a formvar and carbon-coated grid. Although cnidocils were still wet, the grid was placed in a droplet of cold lysis buffer (10 mM cacodylate buffer, pH 7.0, with 10 mM ethylenediaminetetraacetic acid and 0.1% Triton X-100) containing 5% polyethylene glycol (MW 6,000) for 45 minutes. The grid was then placed in another droplet of cold lysis buffer containing 1% polyethylene glycol (MW 6,000) and 1 mM dithioerythritol (DTE) for 40 minutes. The resulting dispersed filaments of cnidocils were fixed with 1% formaldehyde in 10 mM cacodylate buffer for 1 minute and stained with 1% aqueous uranyl acetate for 2030 seconds, air-dried, and observed in the TEM. For immunogold labeling, fixed filaments were treated with the blocking solution as above for 30 minutes, then incubated with anti-nematocilin antibody (1 : 1,000 dilution) for 2 hours at 4°C. After several rinses with 0.1 M cacodylate buffer (pH 7.0), the grids were incubated with Nanogold - goat anti-rabbit IgG for 2 hours, then enhanced with HQ Silver, negatively stained with 1% aqueous uranyl acetate and air-dried before TEM examination.

Electron micrographs of Hydra have shown that the central filament of cnidocils contains longitudinally organized fibers with a characteristic pattern of cross-striations. when sections were stained with immunogold-labeled antibodies, immunogold staining was clearly localized to the central filament in both longitudinal and cross - sections. The peripheral region containing doublet and singlet microtubules did not stain with the anti-nematocilin antibody. Incubation of isolated cnidocils with the reducing agent DTE causes dissociation into individual fibers with a diameter of 34 nm. The central filament could be splayed out into hair-like fibers, which stained strongly with the immunogold-labeled nematocilin antibodies. Hence, it was concluded that nematocilin fibers form the central filament. Both immunofluorescence and immunogold electron microscopy therefore confirmed that nematocilin forms filaments in the central core of the cnidocil.

Nematocilin represents a new member of the intermediate filament superfamily. Two paralogous sequences of nematocilin are present in the Hydra genome: these appear to be the result of recent gene duplication. Comparison of the exonintron structure suggests that the nematocilin genes evolved from the nuclear lamin gene by conserving exons encoding the coiled-coil domains and replacing the C-terminal lamin domains. Molecular phylogenetic analyses also support the hypothesis of a common ancestor between lamin and nematocilin. Comparison of cnidocil structures in different cnidarians indicates that a central filament is present in the cnidocils of several hydrozoan and a cubozoan species but is absent in the cnidocils of anthozoans. A nematocilin homolog is absent in the recently completed genome of the anthozoan Nematostella. Therefore, the evolution of a novel ciliary structure, which provides mechanical rigidity to the sensory cilium during the process of mechanoreception, is associated with the evolution of a novel protein.

Reference:

• Hwang, J. S.; Takaku, Y.; Chapman, J.; Ikeo, K.; David, C. N.; and Gojobori, T.: Cilium evolution: identification of a novel protein, nematocilin, in the mechanosensory cilium of Hydra nematocytes. Mol. Biol. Evol., 25, 2009-17 (2008).
  • Abstract (courtesy of Molecular Biology and Evolution).

More information:

• Nanogold conjugates: catalog information
• Nanogold-antibody probes: instructions and product information
• Nanogold conjugates: technical help
• References: Nanogold post-embedding labeling
• Nanogold references, sorted by application
• Silver enhancers: catalog information
• HQ Silver: instructions and product information
• Silver enhancement references, sorted by application

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

New Grants Extend Medical Imaging Applications with Gold

Nanoprobes has previously developed the first gold nanoparticle X-ray contrast agents for critical angiography and kidney imaging applications. These agents have important advantages over conventional iodine or gadolinium-based contrast agents. They provide enhanced imaging in patients for whom iodine achieves insufficient contrast. They are also excellent for kidney imaging, both because their residence profile allows concentration in the kidneys before clearance, and especially since they do not exhibit the nephrotoxicity properties associated with iodine or gadolinium contrast agents.

A commercial product based on this technology, AuroVist, is available for research and non-clinical applications. Some results obtained with these reagents are shown below.

More information:

• Gold nanoparticles for clinical imaging
• Company information
• News index
• Nanoprobes technology

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Other Recent Publications

Reference:

• Wang, X.; Li, G.; Chen, T.; Yang, M.; Zhang, Z.; Wu, T., and Chen, H.: Polymer-Encapsulated Gold-Nanoparticle Dimers: Facile Preparation and Catalytical Application in Guided Growth of Dimeric ZnO-Nanowires. Nano Lett.,, 8, 2643-2647 (2008).
  • Abstract (courtesy of ACS Publications).

Reference:

• Viens, A.; Harper, F.; Pichard, E.; Comisso, M.; Pierron, G., and Ogryzko, V.: Use of protein biotinylation in vivo for immunoelectron microscopic localization of a specific protein isoform. J. Histochem. Cytochem., 56, 911-919 (2008).
  • Abstract (courtesy of the Journal of Histochemistry and Cytochemistry).

Another method for preparing stable gold nanoparticles was added to the list recently by Aqil and colleagues, reporting in Ultrasonics Sonochemistry: sonoelectrochemistry. Stable, aqueous suspensions of gold nanoparticles were prepared in high yield using a novel one-step, ultrasound assisted electrochemical process. It was found that the addition of either custom polymers or mixtures of commercially available polymers to the electrochemical bath successfully mitigated nanoparticle aggregation, a common problem in sonoelectrochemistry. alpha-Methoxy-omega-mercapto-poly(ethylene oxide) or poly(vinyl pyrrolidone)/polyethylene oxide mixtures produced a coalescence barrier around the gold nanoparticles. The size of the gold nanoparticles could be easily tuned between 5 nm and 35 nm by simple control of the electrochemical parameters - specifically, the deposition time (TON) from 10 ms to 20 ms. The properties of as-prepared gold nanoparticles were compared to the ones of gold colloids prepared by the more conventional wet nanoprecipitation method using chemical reductive agents indicated that their monodispersities and properties were similar. TEM analysis of gold nanoparticles prepared by the sonoelectrochemical method yielded average sizes of 5 1 nm (stabilized by PEO disulfide, E_ON = -1300 mV, TON = 10 ms), 20 4 nm (stabilized by MPEO/PVPr mixtureE_ON = -1300 mV, TON = 20 ms) (gold diameter ) and 35 4 nm (stabilized by PEO disulfide, E_ON = -1300 mV, TON = 20 ms); nanoparticles prepared by chemical methods yielded sizes of 5 2 nm (chemical route stabilized by MPEO) and 5 0.5 nm (stabilized by PEO disulfide).

Reference:

• Aqil, A.; Serwas, H.; Delplancke, J. L.; Jérôme, R.; Jérôme, C., and Canet, L.: Preparation of stable suspensions of gold nanoparticles in water by sonoelectrochemistry. Ultrason. Sonochem., 15, 1055-1061 (2008).
  • Abstract (courtesy of Science Direct).

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .