N A N O P R O B E S     E - N E W S

Vol. 10, No. 7          July 25, 2009

Our existing product, Ni-NTA-Nanogold® has provided submolecular precision for labeling protein subunits for cryoEM. With autometallographic enhancement, it also provides rapid, highly sensitive detection of His-tagged proteins in western blots using our GoldiBlot system.

The new 5 nm Ni-NTA-Gold provides new features, improved performance, and extends NTA-Ni(II) targeting technology to larger gold. This probe, which will be available from August 15, has the same high resolution as Ni-NTA-Nanogold, and the entire probe will still be smaller than an IgG molecule, but the larger gold particles may be clearly visualized by standard TEM without silver or gold enhancement, even in wider views such as thick sections and whole cells: and blot sensitivity will be even higher due to the larger gold.

Features and advantages of 5 nm Ni-NTA-Gold:

• High visibility: The 5 nm gold particle is clearly visualized at TEM resolution without the need for silver or gold enhancement. This simplifies labeling and provides a more monodisperse size range. You may also use this probe for multiple labeling studies in conjunction with gold particles of different sizes, or with silver or gold-enhanced Nanogold.

• Precise 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: we estimate that the distance from the gold particle surface to the His tag is on the order of 1.5 nm. This makes NTA-Ni(II)-Nanogold ideal for localizing sites in protein complexes or other macromolecular assemblies at molecular resolution.

• High solubility and stability: 5 nm Ni-NTA-Gold is prepared using a modified gold particle, using a stable, highly hydrophilic surface functionalization.

• Strong binding: 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.

• High penetration: 5 nm Ni-NTA-Gold is smaller than an unlabeled primary antibody, and can more easily penetrate into specimens and access sterically restricted interior sites, and perturbs the ultrastructure less. In some systems it may be used with stronger fixation or less permeabilization, enabling labeling with better ultrastructural preservation.

• Super sensitivity: the larger gold particle provides higher sensitivity with virtually no background when used to detect His-tagged targets on blots. 10 ng of His-tagged ATF-1 was detected without silver or gold enhancement. Gold enhancement allows the detection of 0.5 ng, with no visible binding to an E Coli. extract control.

Applications of Ni-NTA-Gold probes include:

• High-resolution labeling of proteins, protein complexes or organelles containing recombinant His-tagged proteins for TEM or STEM localization.
• "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.

Product details:

Reference:

• Reddy, V.; Lymar, E.; Hu, M., and Hainfeld, J. F.: 5 nm Gold-Ni-NTA binds His Tags. Microsc. Microanal., 11, (Suppl. 2: Proceedings)(Proceedings of Microscopy and Microanalysis 2005); Price, R.; Kotula, P.; Marko, M.; Scott, J. H.; Vander Voort, G. F.; Nanilova, E.; Mah Lee Ng, M.; Smith, K.; Griffin, P.; Smith, P., and McKernan, S. (Eds.); Cambridge University Press, New York, NY, 1216CD (2005).
  • Paper (HTML) (reproduced courtesy of Cambridge Journals Online).

Functionalized Gold Nanoparticles

If you use gold nanoparticles for nanotechnology or material science applications, you will find our upcoming range of gold nanoparticles novel and useful. We have applied our core technology - the functionalization of gold nanoparticles to give desirable properties - to larger gold particles to give a range of products where you can pick the gold size and the type of solubility behavior you want. The following four products will be available from August 15:

Features and advantages:

• Control of solubility properties: Choose from hydrophobic octane- and dodecanethiol functionalized gold nanoparticles, which have great solubility in organic solvents such as toluene; hydrophilic (1-mercaptoundec-11-yl)tetraethyleneglycol functionalized gold nanoparticles, which dissolve in water and alcohols; and 1-mercapto-(triethyleneglycol) methyl ester functionalized gold nanoparticles which are amphiphilic, dissolving in a variety of solvents including toluene, chloroform, ethyl acetate, acetone, water and alcohols.

• Sized and packaged for nanotechnology and materials applications: our 100 mg size gives you working quantities of product for larger-scale nanomaterials applications or for macro-scale preparations at an affordable and competitive price.

• High stability: We have carefully optimized the ligand chemistry of these particles for stability. The particles will be stable to most environments and the choice of solubility properties lets you select a particle with the interaction properties you need.

Product details:

More information:

• 5 nm Ni-NTA-Gold: our paper from Microscopy and Microanalysis 2005
• Ni-NTA-Nanogold: catalog and general information
• Ni-NTA-Nanogold: product information and instructions
• Technical help: Ni-NTA-Nanogold
• References: Ni-NTA-Nanogold

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Get Close to Your Gold: Linker Lengths

The actual chain configurations for Monomaleimido Nanogold and Mono-Sulfo-NHS-Nanogold are shown below, and the distances calculated, based on the components of the bond lengths that are perpendicular to the gold surface if the cross-linker chain is fully extended.

It should be noted that in many cases the distances are significantly shorter as the gold particle will tend to adsorb to the surface of the conjugate biomolecule; this effect is observed by scanning transmission electron microscopy (STEM) for Nanogold IgG and Fab' conjugates.

If you are labeling a molecule with multiple thiols, it is possible that other reaction mechanisms may contribute to conjugation. Thiols have a very high affinity for gold, and can displace the ligands used in Nanogold and undecagold. Such direct thiol coordination to the gold surface may result in a shorter cross-link length. We have found that conjugation of Fab' antibody fragments occurs both to Nanogold and to larger gold particles stabilized with modified alkylthiols; while this may be helpful for more precise labeling, it may also act to obstruct active sites and change the conformation or properties of the conjugate biomolecule.

If you want to reduce or eliminate the possibility of secondary Nanogold-thiol interactions, we recommend switching the conjugation reaction, introducing a primary aliphatic amine in place of your thiol, and using Mono-Sulfo-N-hydroxysuccinimido Nanogold for the labeling reaction: this will remove the possibility of direct thiol coordination. If you are conducting an amine labeling but wish to eliminate potential interactions with thiols elsewhere in the molecule (such as cysteine residues), block these using N-ethylmaleimide before Nanogold reaction.

Still have questions? Let us advise. Call us at 1-877-447-6266 (US and Canada) or +1 (631) 205-9490, or e-mail us.

More information:

• Guide to Gold Cluster Labeling
• Nanogold labeling reagents: catalog information
• Monomaleimido Nanogold: instructions and product information
• Mono-Sulfo-NHS Nanogold: instructions and product information
• Technical help: Nanogold labeling reagents
• Nanogold labeling references, sorted by application

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Charged Nanogold® Labels Endocytic Compartments in Yeast Cryosections

One such property is ionic charge - positive and negative charge. As shown below, our Positively Charged Nanogold and Negatively Charged Nanogold reagents are prepared by incorporating multiple amines and carboxylic acid groups, respectively, into the Nanogold ligand shell. Positively and negatively charged Nanogold may be used as ionic labels to bind or stain oppositely charged biological targets: for example, we have demonstrated charge-based labeling of oligonucleotides, and Positively Charged Nanogold has been used to trace the yeast and pollen tube endocytic pathways.

These reagents are also potentially important building blocks for nanostructured materials and nanodevices. Decoration of DNA with positively charged Nanogold is a potential method for preparing conductive nanowires, and because they are multiply functionalized, Charged Nanogold molecules may be used for the attachment of multiple conjugate biomolecules to make polyfunctional probes. They can also provide enhanced efficiency in DNA transfection.

Yeast Saccharomyces cerevisiae has been a valuable model organism for the study of the endosomal system of eukaryotic cells; however, until now morphological analyses have been limited because of the lack of specific protein markers and procedures that yield sufficient ultrastructural resolution. Griffith and Reggiori, in their paper in the current Journal of Histochemistry and Cytochemistry, describe an immunoelectron microscopy (IEM) protocol adapted from the Tokuyasu method to prepare cryosections from mildly fixed yeast. This approach allows excellent cell preservation and a unique level of resolution of the yeast morphology. This protocol was combined with specific labeling of various endosomal compartments using Positively Charged Nanogold.

S. cerevisiae cells were grown in a rich medium (yeast extract peptone dexthrose (YPD); 1% yeast extract, 2% peptone, 2% glucose) to exponential phase (optical density (OD)600 5 0.71.0). At this point, ten OD600-unit equivalents of cells were collected by centrifugation at 3000 rpm for 5 minutes, and converted to spheroplasts: first, the cells were resuspended in 5 mL of 100 mM PIPES (pH 9.6) and 10 mM dithiothreitol and incubated at 30°C for 10 minutes. Then, they were collected again by centrifugation and resuspended in 5 mL of YPD medium containing 1 M sorbitol and 5 mg of lytic enzyme, and incubated at 30°C for 30 minutes. The suspension was then centrifuged at 1500 rpm for 5 minutes, and the resulting spheroplasts resuspended in 960 µL of ice-cold YPD medium containing 1 M sorbitol.

30 nmol of Positively Charged Nanogold was suspended in 300 µL of distilled water by extensive vortexing to give a stock solution of 0.1 nmol/µL. Spheroplasts were gently mixed with 4 nmol of Positively Charged Nanogold (40 µL of the stock solution) and placed on ice for 15 minutes. Samples were then transferred to room temperature for 0, 5, 10, 15, 20, and 30 minutes. Nanogold uptake by the spheroplasts was stopped by addition of 1 mL of double-strength fixative (4% (w/v) paraformaldehyde (PFA), 0.4% (v/v) glutaraldehyde (GA), 1 M sorbitol in 0.1 M PHEM buffer (20 mM PIPES, 50 mM HEPES, pH 6.9, 20 mM EGTA, and 4 mM MgCl₂) at room temperature. Tubes were gently inverted several times for 30 minutes. The solution was then centrifuged twice at 6000 rpm for 25 seconds. The fixative was then replaced by fresh standard-strength fixative with 1M sorbitol for an additional two hours at room temperature on a slowly moving rotator.

Samples were then processed for cryosectioning. Cells were resuspended in 1 mL of 0.1 M PHEM buffer and transferred in a 1.5-mL microfuge tube where they were washed three times with the same buffer before adding 12% gelatine dissolved in 0.1 M PHEM buffer at 37°C. This resuspension was then kept at 37°C for 10 minutes to properly infiltrate the clumps of yeast cells. After solidification at 4°C, blocks of about 1 mm³ were trimmed under a dissection microscope at 4°C. These gelatine-embedded blocks were immersed overnight in 2.3 M sucrose in rotating vials at 4°C, then mounted on ultramicrotome specimen holders and frozen by plunging into liquid nitrogen. After trimming to a suitable block shape, ~50 nm ultrathin sections were cut at -120°C on dry diamond knives. Flat ribbons of sections were shifted from the knife-edge with an eyelash and picked up in a wire loop filled with a drop of 1% (w/v) methyl cellulose with 1.15 M sucrose in PBS buffer. Sections were thawed on the pickup droplet and transferred, sections downwards, to Formvar carbon-coated copper grids.

The positively charged Nanogold was then enhanced using HQ Silver for 6 minutes at 24°C. Sections were then stained for 5 minutes with 2% uranyl oxalate acetate (pH 7) at room temperature, and passed over a drop of distilled water to a mixture of 1.8% methyl cellulose and 0.6% uranyl acetate (pH 4) on ice. After 5 minutes the grids were withdrawn, the excess viscous solution drained away, and the sections allowed to dry. Some sections were then immunogold labeled after enhancement.

Nanogold was detected in various morphologically distinct compartments. When spheroplasts were solely kept at 4°C, these particles were exclusively found at the PM. When endocytosis was allowed to occur for 5 minutes, Nanogold was also detected in small vesicles, very likely of endocytic origin. After 10 and 15 minutes, clusters of vesicles and tubules became labeled as well, and those are the yeast EE, and Nanogold was also occasionally observed in the LE/MVBs. Labeling of these structures became much more prominent and uniform after 20 and 30 minutes; and some vacuoles were finally labeled with the Nanogold after 30 minutes. These data are consistent with previous observations, and confirm that Nanogold can be used to label the compartments of the endocytic route as well as demonstrating the high resolution of this method.

The combination of the new IEM protocol and Positively Charged Nanogold labeling produced excellent results when applied for the examination of early and late endosomes, and also the study of mutants with an endosomal trafficking defect. It is also compatible with immunogold labeling of protein markers, and therefore it is appropriate for localization studies of both resident and cargo proteins. This promises to be a valuable tool for scientists using yeast as a model system to investigate the mechanisms of membrane transport, and the biogenesis of the endosomal system.

Reference:

• Griffith, J., and Reggiori, F.: Ultrastructural Analysis of Nanogold-labeled Endocytic Compartments of Yeast Saccharomyces cerevisiae Using a Cryosectioning Procedure. J. Histochem. Cytochem., 57, 801-809 (2009).
  • Abstract (courtesy of the Journal of Histochemistry and Cytochemistry).

Reference for Cryosectioning procedure:

• Griffith, J.; Mari, M.; De Maziere, A., and Reggiori, F.: A cryosectioning procedure for the ultrastructural analysis and the immunogold labelling of yeast Saccharomyces cerevisiae. Traffic, 9, 10601072 (2008).
  • Abstract (courtesy of Wiley Interscience).

More information:

• Charged Nanogold: catalog information
• Positively Charged Nanogold: product information and instructions
• Negatively Charged Nanogold: product information and instructions
• DNA Nanowires: our paper from Microscopy & Microanalysis 2001
• References: Charged Nanogold

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NanoVan and the Genomic Analysis of Streptococcus pneumoniae Bacteriophages

Streptococcus pneumoniae is an important human pathogen, and often carries temperate bacteriophages. To characterize the genetic makeup of prophages associated with clinical strains and to assess the potential roles that they play in the biology and pathogenesis in their host, Romero and group performed comparative genomic analysis of 10 temperate pneumococcal phages. As they demonstrate in their paper in the current issue of the Journal of Bacteriology, electron microscopy was used to confirm the integrity of the genetic material obtained from lysates before sequencing.

In order to examine the lysates, Negative stains, such as our NanoVan (methylamine vanadate) and Nano-W (methylamine tungstate), are particularly useful for studies of virus and protein ultrastructure, and are useful in identifying aberrant protein crystallization in conditions such as Alzheimer's Disease. 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.

Advantages of these reagents:

• 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 and compatibility with other reagents.
• NanoVan is less susceptible to electron beam damage than uranyl acetate.
• Fine grain allows high imaging resolution, useful for image analysis and macromolecule identification.

Crude preparations of bacteriophages were obtained following mitomycin C induction of lysogenic strains. Each strain culture was grown for 8 hours in brain heart infusion broth, then diluted 1:100 in fresh medium. When an optical density at 600 nm of 0.1 to 0.25 was reached, mitomycin C was added to give a final concentration of 100 ng/mL. The culture was then incubated at 37°C until lysis was observed. The lysate was centrifuged for 20 minutes at 3,300 x g at 4°C. The supernatant was then centrifuged at 110,000 x g for 1 hour at 4°C, and the resulting pellet resuspended in 100 µL of ammonium acetate (0.1 M, pH 7.2). Phage preparations were then negatively stained with NanoVan (methylamine vanadate) staining solution on carbon-reinforced, Formvar-coated copper grids (300 mesh). The grid was impregnated with 5 µL of the phage sample and left for 1 minute. Next, 5 µL of negative stain was placed on the grid for 1 minute and washed with the same volume of water for 1 minute. The grids were dried at room temperature for 1 hour, then the samples were observed using the electron microscope (working at 80 kV). Phage DNA was then purified from crude extracts of strain CGSSp6BS73: the pellet obtained from the lysate after mitomycin C induction was resuspended in 10 mM Tris buffer (pH 8.0) and treated with DNase I (1 mg/ml). The material was treated with 50 mM EDTA, 0.5% sodium dodecyl sulfate, and 100 µg/ml proteinase K for 2 hours at 37°C. Finally, DNA was isolated following phenol-chloroform steps and resuspended in 100 µL of of Tris-EDTA buffer (10 mM Tris, 1 mM EDTA, pH 8.0).

Mitomycin C lysates of lysogenic strains OXC141, CGSSp3BS71, CGSSp6BS73, CGSSp9BS68, CGSSp11BS70, CGSSp14BS69, CGSSp18BS74, CGSSp19BS75, CGSSp23BS72, and 23F were observed with an electron microscope. That from strain CGSSp23BS68 contained only capsids, while CGSSp9BS72 did not display any phage-like particles. However, in all the other preparations, phage particles were observed showing a Siphoviridae morphology. The sizes of the tails and heads observed were homogeneous (approximately 50 x 50 nm for the heads, and 200 nm for the tails).

Upon genomic analysis, it was found that all of the genomes are organized into five major gene clusters: lysogeny, replication, packaging, morphogenesis, and lysis clusters. Of the phage particles observed that showed a Siphoviridae morphology. The only genes that are well conserved in all the genomes studied were those involved in the integration and the lysis of the host, in addition to two genes, of unknown function, within the replication module. A high percentage of the open reading frames contained no similarities to any sequences catalogued in public databases, but genes homologous to known phage virulence genes, including the pblB gene of Streptococcus mitis and the vapE gene of Dichelobacter nodosus, were also identified. In addition, the phage øSpn_6 was found to contain an toxin-antitoxin system: this represents the first time that an addition system has been identified in a pneumophage. Overall, the temperate pneumophages were found to contain a diverse set of genes, with various levels of similarity among them.

Reference:

• Romero, P.; Croucher, N. J.; Hiller, N. L.; Hu, F. Z.; Ehrlich, G. D.; Bentley, S. D.; García, E., and Mitchell, T. J.: Comparative genomic analysis of ten Streptococcus pneumoniae temperate bacteriophages. J. Bacteriol., 191, 4854-4862 (2009).
  • Abstract (courtesy of the Journal of Bacteriology).

More information:

• Catalog information: Negative stains
• NanoVan: Product information and instructions
• Nano-W: Product information and instructions
• NanoVan: our presentation at Microscopy and Microanalysis 1994
• Technical help: Negative staining
• Negative staining: Other references

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See us at Microscopy & Microanalysis 2009

The exposition will be open from Monday, July 27 through Thursday, July 30; it opens at 12:00 noon on Monday and closes at 5:00 PM, then opens from 9:30 AM to 5:00 PM Tuesday and Wendesday, and from 9:30 AM to 3:00 PM on Thursday. We will be in booth 555; check our location in the floor plan: we will be just across the aisle from Omniprobe, and one aisle over from the MSA Megabooth.

More information:

• Microscopy and Microanalysis 2009 - Information
• Microscopy and Microanalysis 2009 - Scientific Program Search
• Complete Program (courtesy of Cambridge University Press)
• Nanoprobes: about us - company information
• Our technology
• News

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Other Recent Publications

By applying double labeling of CFs with anterograde tracer and VGluT2 antibody to light and electron microscopic analyses, we have disclosed that pericellular nests do represent multiple CF innervation of PCs, and that a single main CF extends progressively toward distal spiny branchlets with other surplus CFs remaining around the soma and the basal portion of dendrites. For electron microscopy, BDA-labeled sections were incubated overnight with guinea pig VGluT2 antibody and avidin-biotin-peroxidase complex. Then, sections were incubated with Nanogold-conjugated anti-guinea pig antibody, diluted 1:200 for 3 hours. The Nanogold for VGluT2 was enhanced with HQ Silver, and then BDA was visualized using DAB. Serial ultrathin sections were prepared in the plane parallel to the pial surface. 3D reconstructed images were built using a free software package (Reconstruction, available at Synapse Web).

Combination of microscopic and electrophysiological studies confirmed that competition among multiple CFs occurs on the soma before CFs form synapses along dendrites. In most PCs, the single CF that has been functionally strengthened (the "winner" CF) undergoes translocation to dendrites while keeping its synapses on the soma. Synapses of the weaker CFs (the "loser" CFs) remain around the soma and form "pericellular nests" with synapses of the winner CFs. Most perisomatic synapses are then eliminated non-selectively by P15. Thus, our results suggest that the selective translocation of the winner CF to dendrites in each PC determines the single CF that survives subsequent synapse elimination and persistently innervates the PC.

Reference:

• Hashimoto, K.; Ichikawa, R.; Kitamura, K.; Watanabe, M., and Kano, M.: Translocation of a "winner" climbing fiber to the Purkinje cell dendrite and subsequent elimination of "losers" from the soma in developing cerebellum. Neuron, 63, 106-118 (2009).
  • Abstract (courtesy of Neuron).

Nanogold was used to label PV or CR, while D1 or D5 was labeled with DAB. Tissue sections were thawed and incubated with blocking serum (3% normal goat serum, 1% bovine serum albumin, 0.1% glycine and lysine, and 0.5% fish gelatin made in phosphate-buffered saline) for one hour at room temperature. Sections were incubated overnight in a mixture of primary antibodies (rat anti-D1 diluted 1:500 or rabbit anti-D5, 1:500; and mouse anti-PV, 1:10,000 or mouse anti-CR, 1:10,000), then incubated overnight in a cocktail of secondary antisera (biotinylated donkey anti-rat, 1:200; or biotinylated goat anti-rabbit, 1:200; and Nanogold goat anti-mouse at 1:200). Sections were then postfixed in 2% glutaraldehyde for 20 minutes and enhanced for 3-5 minutes with HQ Silver. After incubation in ABC reagent for one hour at room temperature, the sections were reacted with DAB and 0.3% H₂O₂. The sections were then osmicated in 0.5% OsO₄ for 10 minutes, dehydrated in ethanol and propylene oxide, then flat-embedded in Durcupan resin. Control sections, in which one of the two primary immunoreagents was omitted, showed no evidence either for nonspecific deposition of gold particles (except for cell nuclei, which were found to nonspecifically interact with gold-labeled antibodies) or for nonspecific deposition of DAB onto previously developed gold particles.

Each antibody produced labeling only when incubated with the corresponding secondary antibody, indicating that spurious immunolabeling is unlikely in the double-labeling conditions. D1 was found to be the predominant D1R subtype in PV interneurons, and was found mainly in dendrites. In contrast, D5 was the predominant D1R subtype in CR interneurons, and was found mainly in dendrites. By integrating these findings with electrophysiological data, the authors proposed a circuitry model as a framework for understanding the inverted-U relationship between dopamine stimulation of D1R and WM performance.

Reference:

• Glausier, J. R.; Khan Z. U., and Muly, E. C. Dopamine D1 and D5 receptors are localized to discrete populations of interneurons in primate prefrontal cortex. Cereb. Cortex, 19, 1820-1834 (2009).
  • Abstract (courtesy of Oxford Journals).

In this month's Nano Letters, Wang and colleagues experimentally demonstrate the enhanced propulsion of 250 nm gold nanoparticles by surface plasmon polaritons (SPPs). Three dimensional finite difference time domain (FDTD) simulations indicate considerably enhanced optical forces due to the field enhancement provided by SPPs and the near-field coupling between the gold particles and the film. This coupling is an important part of the enhanced propulsion phenomenon. Finally, the measured optical force is compared with that predicted by FDTD simulations and proven to be reasonable. These observations provide more insight into the optical manipulation of gold nanoparticles as a tool for their applications in nanotechnology.

Reference:

• Wang, K.; Schonbrun, E., and Crozier, K. B.: Propulsion of gold nanoparticles with surface plasmon polaritons: evidence of enhanced optical force from near-field coupling between gold particle and gold film. Nano Lett., 9, 2623-2629 (2009).
  • Abstract (courtesy of ACS Publications).

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