N A N O P R O B E S     E - N E W S

Vol. 10, No. 2          February 28, 2009

Elizabeth Schröder-Reiter, Gerhard Wanner and group have contributed several advances to the SEM use of Nanogold and FluoroNanogold. In 2006, they reported the first 3D SEM analysis of a nucleolus organizing region (NOR) with an atypical peg-like terminal constriction on metaphase plant chromosomes. Improvements in signal localization, labeling efficiency, and structural preservation in the SEM ISH procedure allowed 3D SEM analysis of the NOR structure and rDNA distribution for the first time. In a new paper in the Journal of Structural Biology, they describe the use of focused ion beam (FIB) milling in combination with field emission scanning electron microscopy (FESEM) for investigations of metaphase barley chromosomes, and report new insight into the chromatin packaging in the chromosome interior and 3D distribution of histone variants in the centromeric region.

In combination with immunogold labeling, the centromere-correlated distribution of histone variants (phosphorylated histone H3, CENH3) were investigated in three dimensions. Divisions of root-tip cells of sprouting barley seeds (Hordeum vulgare Steffi, 2n = 14) were synchronized, arrested at metaphase, and fixed with 3:1 (v/v) ethanol:acetic acid. Chromosomes were isolated and dropped onto glass slides using the Drop/Cryo method. The chromosome preparations were washed in phosphate buffered saline (PBS) buffer, blocked in 1% bovine serum albumin with 0.1% Tween 20 in PBS for 30 minutes, then incubated with the respective primary antibody (diluted in blocking solution) for 1 hour (1:250 rabbit anti-serine 10 phosphorylated histone H3, or 1:300 rabbit anti-OsCENH3). After washing, specimens were incubated with Nanogold-Fab' Goat anti-Rabbit fragments for 1 hour. Specimens were washed and post-fixed with 2.5% glutardialdehyde in PBS, then silver enhanced using HQ Silver. For DNA staining, chromosomes are incubated for 30 minutes at room temperature with Platinum Blue ([CH₃CN]₂Pt oligomer, 10 mM, pH 7.2), then washed with distilled water. Whole mount chromosomes were sectioned with FIB with thicknesses in the range of 720 nm, resulting in up to 2000 sections, which allow high resolution three-dimensional reconstruction.

For the first time, the chromosome interior could be shown to be characterized by a network of interconnected cavities, with openings to the chromosome surface. In combination with immunogold labeling, the centromere-correlated distribution of histone variants (phosphorylated histone H3, CENH3) could be investigated with FIB in three dimensions. FIB milling overcomes the limitations of classical SEM analysis of whole mount chromosomes with back-scattered electrons, which require higher accelerating voltages, such as faint and blurred interior signals. From within the chromosome, even very small labels in the range of 10 nm could be precisely visualized, which allowed direct quantification of marker molecules in a three-dimensional context. Distribution of DNA in the chromosome interior could be directly analyzed after staining with a DNA-specific platinorganic compound Platinum Blue. Higher resolution visualization of DNA distribution could be performed by preparation of FIB lamellae with the in situ lift-out technique followed by investigation in dark field with a scanning transmission electron detector (STEM) at 30 kV.

GoldEnhance has several advantages for different applications in addition to its enhanced backscatter detection for SEM:

• Gold enhancement may safely be used before any strength osmium tetroxide - it is not etched.
• May be used in physiological buffers (including chlorides, which precipitate silver, but not gold).
• The metallographic reaction is less pH sensitive than that of silver.
• Gold gives a much stronger backscatter signal than silver.
• GoldEnhance is near neutral pH for best ultrastructural preservation.
• Low viscosity, so the components may be dispensed and mixed easily and accurately.

Together with Nanogold and FluoroNanogold, these reagents provide a set of research tools for many difficult structural and microscopical problems.

References:

• Hermann, R.; Walther, P., and Muller, M.: High precision immuno-scanning electron microscopy using Fab fragments coupled to ultra-small colloidal gold. J. Struct. Biol., 107, 38-47 (1991).
  • Abstract (courtesy of Science Direct).

• Hermann, R.; Walther, P., and Muller, M.: Immunogold labeling in scanning electron microscopy. Histochem. Cell Biol., 106, 31-39 (1996).
  • Abstract (courtesy of SpringerLink).

• Schröder-Reiter, E.; Pérez-Willard, F.; Zeile, U., and Wanner, G.: Focused ion beam (FIB) combined with high resolution scanning electron microscopy: a promising tool for 3D analysis of chromosome architecture. J. Struct. Biol., 165, 97-106 (2009).
  • Abstract (courtesy of Science Direct).

More information:

• Nanogold conjugates: general and catalog information
• Nanogold-antibody conjugates: product information and instructions
• References: Nanogold SEM labeling
• Nanogold conjugates: technical help
• HQ Silver: product information and instructions
• Gold enhancement: catalog and general information
• Gold enhancement: technical help

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Nanogold® Enables Versatile Peptide Labeling

Label at cysteines with Monomaleimido Nanogold®

Monomaleimido Nanogold reacts selectively with thiol (-SH) groups. Use this reagent to label at cysteine residues; alternatively, you can use 2-iminothiolane (Traut's reagent) to convert primary amino- groups to longer-chain thiols if necessary. Thiols are labeled under mild conditions, at pH values between 6 and 7. Typically, Monomaleimido Nanogold is reacted with the peptide overnight at 4°C, sometimes after a brief period (30 minutes to one hour) of gentle agitation, then the conjugate product is isolated next day. If you plan to use this reagent, you should check to make sure that the cysteine is reduced and available for labeling: often, cysteines are oxidized to form disulfide bridges which must be carefully reduced before labeling.

Gregori and co-workers provided an example of cysteine labeling: they labeled a modified tau beta-amyloid peptide containing a cysteine residue with Monomaleimido Nanogold, then used the labeled 39-amino acid peptide to investigate the binding interaction with the 20S proteasome by STEM microscopy. Because of the small size of the labeled peptide, they were able to map binding sites inside the proteasome with high resolution, as shown below.

Reference:

• Gregori, L., Hainfeld, J. F., Simon, M. N., and Goldgaber, D. Binding of amyloid beta protein to the 20S proteasome. J. Biol. Chem., 272, 58-62 (1997).
  • Abstract (courtesy of Science Direct).

Label at lysines or N-terminal amines with Mono-Sulfo-NHS-Nanogold®

Mono-Sulfo-NHS-Nanogold® reacts with primary aliphatic amines: it therefore labels specifically at the N-terminal amine or at a lysine residue in the chain (labeling at the N-terminal is usually slightly preferred, both because it is more accessible and because the N-terminal amine usually has a slightly greater reactivity). This approach is useful because all peptides have at least one reactive site.

Segond von Banchet and Heppelmann used Mono-Sulfo-NHS-Nanogold® to label the bioactive peptide, Substance P (SP); this has only 11 amino acids and a molecular weight of 1,517 Daltons. Careful controls showed that the Nanogold-labeled SP behaved similarly to the commonly used Iodine-125-labeled SP. Specific binding to target proteins on SDS-PAGE blots could be competed off with excess unlabeled SP; binding was also inhibited by a specific NK1-receptor antagonist, but not by neurokinin A, which does not target the NK1 receptor, as shown below. Nanogold alone led to no specific staining.

Reference:

• Segond von Banchet, G., and Heppelmann, B.: Non-radioactive localization of substance P binding sites in rat brain and spinal cord using peptides labeled with 1.4-nm gold particles. J. Histochem. Cytochem., 43 821-827 (1995).
  • Abstract (courtesy of the Journal of Histochemistry and Cytochemistry).
  • Reprint (PDF) (courtesy of the Journal of Histochemistry and Cytochemistry).

Jones and group have described the preparation and use of Nanogold-labeled alpha-bungarotoxin. The conjugate was prepared by using Mono-Sulfo-NHS-Nanogold to label the toxin; conjugation was carried our using standard procedures given in our product instructions, and the product was purified by gel filtration. In order to compare the relative dimensions of alpha-Bgt and Nanogold, the physical size of alpha-Bgt was calculated to using a modeling package; it was shown to be 4.5 times larger than the gold core of the Nanogold particle. Displacement assays showed that alpha-Bgt binding was preserved in the Nanogold conjugate: Ki values are 7.8 and 4.2 nM for gold conjugated alpha-Bgt and unconjugated alpha-Bgt, respectively.

Reference:

• Jones; I. W.; Barik, J.; O' Neill, M. J., and Wonnacott, S.: Alpha bungarotoxin-1.4 nm gold: a novel conjugate for visualising the precise subcellular distribution of alpha 7* nicotinic acetylcholine receptors. J. Neurosci. Methods, 134, 65-74 (2004).
  • Abstract (courtesy of Science Direct).

Label at C-terminal carboxyls with Monoamino Nanogold®

If the only functional group I have is a carboxylic acid, what should you do? You can still label with Nanogold using Monoamino Nanogold®. To carry out the labeling reaction, you need to convert the carboxylic acid group to a reactive ester, which will then react with the amine - the same reaction that occurs when Mono-Sulfo-NHS-Nanogold is used to label an amine. Appropriate reaction schemes are shown below:

The reaction used in peptide synthesis usually works well - react the carboxylic acid with EDC (1-Ethyl-3- [3-dimethylaminopropyl] carbodiimide Hydrochloride) and Sulfo-NHS to convert it to a reactive Sulfo-N-hydroxysuccinimide ester. You can purchase EDC from a number of sources; its use is described by Pierce.

EDC reacts with a carboxyl group on the molecule to be labeled, forming an amine-reactive O-acylisourea intermediate. This intermediate could then react with Monoamino Nanogold; however, it is also susceptible to hydrolysis, making it unstable and short-lived in aqueous solution. The addition of Sulfo-NHS (5 mM) stabilizes the amine-reactive intermediate by converting it to an amine-reactive Sulfo-NHS ester, thus increasing the efficiency of EDC-mediated coupling reactions. The amine-reactive Sulfo-NHS ester intermediate has sufficient stability to permit two-step cross-linking procedures, which allow the carboxyl groups on one protein to remain unaltered.

Reference:

• Staros, J. V.; Wright, R. W., and Swingle, D. M.: Enhancement by N-hydroxysulfosuccinimide of water-soluble carbodiimide-mediated coupling reactions. Anal. Biochem., 156, 220-222 (1986).
  • Abstract (courtesy of PubMed (Medline)).

Another reagent that works well in non-aqueous systems is 1,1-carbonyl-diimidazole (CDI). The molecule to be labeled should be dissolved in a small amount of the organic solvent and a small (5-fold to 10-fold) excess of CDA added; the pH is then raised to 7.5 or higher by the addition of aqueous reaction buffer, and the Monoamino Nanogold added.

Reference:

• Staab, H. A., and Rohr; W.; Newer Methods Prep. Org. Chem., 5, 61 (1968).

More information:

• Special Report: Nanogold-peptide labeling
• Guide to Gold Cluster Labeling
• Nanogold labeling reagents: catalog information
• Monomaleimido Nanogold: Product information and instructions
• Mono-Sulfo-NHS-Nanogold: Product information and instructions
• Monoamino Nanogold: Product information and instructions
• Technical Help Nanogold labeling reagents
• References by Application - Nanogold Labeling

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Neuropeptide Y, Hippocampus-Dependent Seizures, and Nanogold®

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

Together with HQ Silver, this provides the ideal combination of features to produce high-quality staining:

• The only commercial silver enhancer to feature a protective colloid for highest size uniformity and morphologically consistent enhancement.
• Near neutral pH and low ionic strength ensure maximum specimen integrity.
• High proportion of gold particles are enlarged.
• Highly selective reaction with low background.

This month's contribution to this field comes from Ledoux and co-workers: in their paper in the Journal of Neuroscience, they describe the use of Nanogold labeling with silver enhancement to help uncover the mechanism by which neuropeptide Y controls hippocampus-dependent seizures. In about one-third of women with epilepsy, the seizures show a catamenial pattern: they fluctuate with the menstrual cycle. Catamenial seizures occur more frequently when the ratio of circulating estradiol to progesterone is high, which suggests that the convulsions may be promoted by estradiol. The authors used adult female rats to test how estradiol-induced suppression of GABAergic inhibition in the hippocampus affects behavioral seizures induced by kainic acid, which produces highly stereotypical seizures, and light and electron microscopy to monitor the effects upon the amount and distribution of some of the key molecules.

Electron microscopic immunocytochemistry with enzymatic DAB was used in nonseizure animals to localize and quantify neuropeptide Y immunoreactivity (IR) at the ultrastructural level. Rats were deeply anesthetized with sodium pentobarbital and perfused with heparinized saline, followed by 3% paraformaldehyde/0.5% glutaraldehyde in phosphate buffer (PB). Brains were removed, blocked to contain the dorsal hippocampus, and postfixed overnight at 4°C. Blocks were then rinsed and sectioned (50 µm) through the dorsal hippocampus and treated with 1% sodium borohydride and 1% hydrogen peroxide, followed by a blocking solution containing 3% bovine serum albumin (BSA), 0.3% dimethylsulfoxide (DMSO), and 5% normal serum in Tris-buffered saline (TBS) at pH 7.4. After rinsing, sections were incubated in primary antiserum to NPY (rabbit polyclonal, 5.0 µg/mL) in 2% BSA, 0.3% DMSO, and 1% normal serum in TBS at 4µC for 48 hours. Sections were then rinsed and incubated with biotinylated anti-rabbit IgG (1:800) followed by an avidin biotin horseradish peroxidase complex (1:100) in TBS. NPY-IR was visualized with a DAB reaction. Sections were stained with 1% osmium tetroxide, then flat-embedded in Eponate resin and blocked to contain CA1, then mounted for thin sectioning. Short series of ultrathin (~80 nm) sections were cut and collected on formvar-coated slot grids, and stained with 3% uranyl acetate and 2.66% Reynolds lead citrate.

Another group of rats was used for electron microscopic visualization of estrogen receptor-alpha (ERalpha), to investigate whether large dense-core vesicles (LDCVs) contain ERalpha-IR. Tissue preparation was as described above, except that hydrogen peroxide pretreatment was omitted. Sections were incubated in primary antiserum to ERalpha (MC-20 rabbit polyclonal, 0.5 µg/mL) followed by Nanogold anti-Rabbit IgG (1:50 dilution) followed by enhancement with HQ-silver. Controls included omission of primary antiserum, and showed no nonspecific staining. The MC-20 antiserum used for ERalpha labeling does not cross-react with ERbeta, and recognizes a single band of the appropriate size on Western blots from hippocampus; all staining was eliminated by preadsorption with a blocking peptide. Because lots of antisera can vary, each lot of MC-20 was verified by Western blot before use.

As expected, a 24 hour estradiol treatment increases susceptibility to initiation of KA-induced seizures, but it was also observed that estradiol has a mitigating effect, reducing seizure severity once seizures have begun. Additional analyses showed that the decrease in seizure severity was attributable to greater release of the anticonvulsant neuropeptide, neuropeptide Y (NPY). First, blocking hippocampal NPY during seizures eliminated the estradiol-induced decrease in seizure duration. Second, light and electron microscopic studies indicated that estradiol increases the potentially releasable pool of NPY in inhibitory presynaptic boutons and facilitates the release of NPY from inhibitory boutons during seizures. Finally, the presence of estrogen receptor-alpha on large dense-core vesicles (LDCVs) in the hippocampus suggests that estradiol could facilitate neuropeptide release by acting directly on LDCVs themselves. Understanding how estradiol regulates NPY-containing LDCVs could identify potential molecular targets for novel anticonvulsant therapies.

Reference:

• Ledoux, V. A.; Smejkalova, T.; May, R. M.; Cooke, B. M, Woolley, C. S.: Estradiol facilitates the release of neuropeptide Y to suppress hippocampus-dependent seizures. J. Neurosci., 29, 1457-1468 (2009).
  • Abstract (courtesy of the Journal of Neuroscience).

More information:

• Special feature: pre-embedding labeling with HQ Silver enhancement: electron microscopy with Nanogold
• Nanogold-antibodies: general information and catalog
• Nanogold-antibodies: instructions and product information
• Silver enhancement: general information and catalog
• HQ Silver: product information and instructions
• Nanogold conjugates: technical help
• Silver enhancement: technical help
• Nanogold pre-embedding electron microscopy: other references

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Nanogold® Helps Reveal Siliconization Mechanism

Electron immunogold labeling for TEM [transmission electron microscopy] analysis was performed with tissue samples that had been treated in glutaraldehyde/paraformaldehyde. tissue samples treated in 0.1% glutaraldehyde / 3% paraformaldehyde buffered in 0.1 M phosphate buffer at pH 7.4; after 2 h, the material was dehydrated in ethanol and embedded in LR-White Resin. 60 nm slices were cut, and blocked with 5% bovine serum albumin (BSA) in phosphate-buffered saline (PBS), then incubated with the primary antibody PoAb-aSilic (1:1000) for 12 hours at 4°C. For the detection of the antigenantibody complexes, Nanogold anti-Rabbit IgG was applied, and after washing, silver enhancement was conducted using the Danscher procedure. In controls, pre-immune serum was used; in these sections no antigenantibody could be detected.

Microscopic examination showed that the surface of the spicules have, towards the tip, serrated relief structures that are consistent in size and form with the protrusions on the surface of the spicules. These protrusions fit into the collagen net that surrounds the spicules. The widths of the individual lamellae do not show any pronounced size tendency. Apical elongation of the spicule proceeds by piling up cone-like structural units formed from silica. As a support of the assumption that in the extracellular space silicatein(-like) molecules exist that associate with the external surface of the respective spicule immunogold electron microscopic analyses were performed. With the primmorph system from Suberites domuncula, it was demonstrated that silicatein(-like) molecules assemble as string- and net-like arrangements around the spicules. At their tips, these silicatein(-like) molecules are initially stacked, but later they are also organized into net-like structures. Silicatein(-like) molecules were also extracted from the giant basal spicule of Monorhaphis. Applying the SDSPAGE technique revealed that silicatein molecules associate to form dimers and trimers. By scanning electron microscopy, the formation of higher complexes (filaments) from silicatein(-like) molecules was visualized. In the presence of ortho-silicate, these filaments became covered with 3060 nm long small rod-like or cuboid particles of silica.

Structural (SEM analyses) and functional data (enzymic reactions) on the 25 kDa protein in Monorhaphis, together with the cloning information from the hexactinellid Crateromorpha meyeri, indicate that an enzymatically active silicatein exists also in spicules from hexactinellids which islike in demospongesinvolved in bio-silica formation. It was concluded that the apical elongation of the spicules of Monorhaphis proceeds by piling up cone-like silica structural units: the synthesis of these units is mediated by silicatein(-like) molecules.

Reference:

• Wang X.; Boreiko A.; Schlossmacher U.; Brandt D.; Schröder H. C.; Li J.; Kaandorp J. A.; Götz H.; Duschner H, Müller W. E.: Axial growth of hexactinellid spicules: formation of cone-like structural units in the giant basal spicules of the hexactinellid Monorhaphis. J. Struct. Biol., 164, 270-280 (2008).
  • Abstract (courtesy of Science direct).

Reference for specimen preparation and labeling procedure:

• Müller, W. E. G.; Rothenberger, M.; Boreiko, A.; Tremel, W.; Reiber, A., and Schröder, H. C. Formation of siliceous spicules in the marine demosponge Suberites domuncula. Cell Tissue Res., 321, 285297 (2005).
  • Abstract (courtesy of SpringerLink).

More information:

• Nanogold-antibodies: general information and catalog
• Nanogold-antibodies: instructions and product information
• Nanogold conjugates: technical help
• Nanogold labeling references, sorted by application

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Gold Nanoparticles, and When to Order from a Distributor

If you are ordering from outside the United States, there are several important benefits to ordering through one of our distributors for your region. Our distributors are familiar with import procedures and requirements, as well as distribution within your region, it will save you time and possibly expense with customs and import duties to order through a distributor who knows how the system works in your country. Check our distributor list for a distributor in your region.

More information:

• International Distributors
• Nanoprobes: news and press releases
• About Us: company information
• Our story: company history

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Other Recent Publications

Reference:

• Hosta L.; Pla-Roca M.; Arbiol J.; López-Iglesias C.; Samitier J.; Cruz L. J.; Kogan M. J, and Albericio F.: Conjugation of Kahalalide F with gold nanoparticles to enhance in vitro antitumoral activity. Bioconjug. Chem., 20, 138-146 (2009).
  • Abstract (courtesy of ACS Publications).

Reference:

• Perni, S.; Piccirillo, C.; Pratten, J.; Prokopovich, P.; Chrzanowski, W.; Parkin, I. P., Wilson, M.: The antimicrobial properties of light-activated polymers containing methylene blue and gold nanoparticles. Biomaterials, 30, 89-93 (2009).
  • Abstract (courtesy of Science Direct).
  • Reprint (PDF) (courtesy of Science Direct).

Because it is easily functionalized and conjugated, and confers protection in many environments, gold is attractive as a coating for other types of nanoparticles, and a recent Chemistry of Materials paper from Goon and co-workers describes one such preparation: magnetic nanoparticles with a gold shell. An aqueous synthesis of composite 50-150 nm magnetite-gold core-shell nanoparticles is presented, which provides the ability to engineer the coverage of gold on the magnetite particle surface. This method utilizes polyethyleneimine (PEI) for the dual functions of attaching 2 nm gold nanoparticle seeds onto magnetite particles, and also to prevent the formation of large aggregates. In this method, the surface of the magnetite particles is saturated with gold seeds: these are then 'developed' by further treatment with a tetrachloroaurate-based gold development solution to form magnetically responsive core-shell particles, which also exhibit surface plasmon resonance. The gold-shell formation process was characterized using transmission electron microscopy, X-ray photoelectron spectroscopy, energy-dispersive spectroscopy, and inductively coupled plasma optical emission spectroscopy. Dynamic light scattering studies also showed that PEI coating of synthesized particles protected them against aggregation. The ability of the gold shell to protect the magnetite cores was tested by subjecting the particles to a magnetite-specific dissolution procedure. Elemental analysis of dissolved species revealed that the gold coating of magnetite cores imparts remarkable resistance to iron dissolution. The ability to engineer gold coverage on particle surfaces allows for controlled biofunctionalization, whereas their resistance to dissolution ensures applicability in harsh environments.

Reference:

• Goon, I. Y.; Lai, L. M. H.; Lim, M.; Munroe, P.; Gooding, J. J., and Amal, R.: Fabrication and Dispersion of Gold-Shell-Protected Magnetite Nanoparticles: Systematic Control Using Polyethyleneimine. Chem. Mater., 21, 673-681 (2009).
  • Abstract (courtesy of ACS Publications).

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