N A N O P R O B E S     E - N E W S

Vol. 7, No. 7          July 12, 2006

Updated: July 12, 2006

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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Correlative XRF and Immunofluorescence with FluoroNanogold

Synchrotron-based X-ray fluorescence microscopy (microXRF) provides high-resolution localization of a wide range of biologically relevant elements, including gold and other metals, in tissues and cells. When combined with fluorescence microscopy, it enables correlation of the cellular distribution of a target with the ultrastructural mapping of many different elements, raising the possibility of ultrastructural labeling of multiple sites distinguished by different elemental labels in a similar manner to that described previously using electron spectroscopic imaging.

FluoroNanogold is a unique immunoprobe, containing both a fluorophore and the 1.4 nm Nanogold® particle, which may be visualized by both fluorescence microscopy and, as McRae and colleagues report in the current Journal of Structural Biology, X-ray fluorescence microscopy. The introduction of Alexa Fluor®* 488 and 594 FluoroNanogold conjugates, which may be observed by fluorescence microscopy using the same filter sets used for fluorescein and Texas Red conjugates respectively, let you differentiate a second FluoroNanogold-labeled target from a feature labeled with fluorescein, Alexa Fluor® 488, green fluorescent protein, or other green fluorophores. Alexa Fluor®* FluoroNanogold probes offer superior performance:

  • Increased fluorescence brightness and higher quantum yield.
  • Improved solubility: lower background signal and higher signal-to-noise ratios.
  • Fluorescence remains high and consistent across a wider pH range.

An example of the fluorescence labeling that may be obtained with these probes is shown below:

[Alexa Fluor® 488 FluoroNanogold: structure and fluorescence staining] (48k)]

Left: Structure of Alexa Fluor® 488 FluoroNanogold - Fab', showing covalent attachment of components. Right: Fluorescent staining obtained using Alexa Fluor 488 FluoroNanogold as a tertiary probe. The specimen is a slide from the NOVA Lite ANA HEp-2 test, an indirect immunofluorescent test system for the screening and semi-quantitative determination of anti-nuclear antibodies (ANA) in human serum, stained using positive pattern control human sera, a Mouse anti-Human secondary antibody, and Alexa Fluor 488 FluoroNanogold tertiary probe. Specimens were washed with PBS (30 minutes) between each step, then blocked by the addition of 7% nonfat dried milk to the tertiary antibody solution (original magnification 400 X).

McRae and group obtained microXRF elemental maps with well-defined subcellular resolution by growing mouse fibroblast cells directly on formvar-carbon coated electron microscopy grids. NIH 3T3 mouse cells (adherent fibroblasts) were cultured in Dulbeccos modified Eagles medium (DMEM) containing 10% bovine serum, supplemented with 200 mM L-glutamine. Cells were grown to 5080% confluency either on formvar-carbon coated 200 mesh gold-grids or on silicon nitride windows pretreated for 30 minutes with 0.01% poly-L-lysine solution. The cells were fixed at room temperature for 30 minutes with pre-warmed (37°C) 3.7% paraformaldehyde freshly prepared in phosphate-buffered saline (PBS; pH 7.2), then permeabilized with 0.2% Triton X-100 in PBS (pH 7.2) for 10 minutes, then incubated for 1 hour in blocking buffer. The cells were incubated with either mouse anti-GS28 IgG1 (cis-Golgi marker; 1:300 dilution), or anti-OxPhos complex V IgG1 (mitochondrial marker; 1:300 dilution) for 1 hour, washed thoroughly with 0.05% Tween-20 in PBS to remove unbound antibodies, then incubated with Alexa Fluor 488 FluoroNanogold anti-mouse secondary antibody (1:10 dilution) for 1 hour. The cells were then washed again with Tween-20 to remove unbound antibodies. Optical fluorescence micrographs were acquired by mounting the silicon nitride windows onto slides, using PBS as mounting medium, and imaged with an inverted fluorescence microscope equipped with a standard filter set (FITC).

Immediately after fluorescence imaging, the cells were rinsed quickly with PBS then twice with isotonic ammonium acetate (0.1M) prepared in ultrapure water (18MOhms), and dried in air overnight. Scanning X-ray fluorescence microscopy was performed at the 2-IDD beamline of the Advanced Photon Source at Argonne National Laboratory (IL, USA). The grids were placed onto a kinematic specimen holder suitable for both optical and X-ray fluorescence microscopy, which was mounted on a light microscope. Target cells imaged previously by standard fluorescence microscopy were located on the grid relative to a reference point using a high spatial resolution motorized x/y stage; coordinates were determined and used to precisely locate the target cell once the grid was transferred to the microprobe. A Fresnel zone plate was used to focus the monochromatic X-ray beam from an undulator source to a spot size of 0.2 x 0.2 µm2 on the specimen. An incident photon energy of 11.95keV was chosen to ensure excitation of the Lalpha line of gold, and the sample was raster scanned through the beam at 298K under a helium atmosphere. Pixel step size was set to 0.2 µm and the entire X-ray spectrum recorded at each pixel using an energy dispersive germanium detector.

Elemental maps were created by spectral filtering, using spectral regions of interest matched to characteristic X-ray emission lines to determine the fluorescence signal for each element. To improve accuracy and reduce crosstalk between overlapping fluorescence lines, such as the Zn Kbeta (ED9572 eV) and Au Lalpha (E1D9713 eV, E2D9628 eV), spectra acquired at every pixel were fitted individually using an adapted version of the SNIP algorithm for background estimation, and modified gaussians for peak fitting. Calibration to elemental area densities (µg/cm2) was performed by comparison of X-ray fluorescence signal strength from the sample to fluorescence from thin film standards NBS-1832 and NBS-1833 from the National Bureau of Standards (NBS/NIST, Gaithersburg, MD) using MAPS software. Elemental content was calculated by fitting the integrated spectra of the acquired fluorescence datasets, then comparing fitted fluorescence signal strength to that resulting from fitting of NBS 1832/33 standard spectra.

This method provided two-dimensional maps with submicron resolution for gold, as well as for most biologically relevant elements. MicroXRF proved to be sufficiently sensitive to image the location and structural details of the gold-labeled organelles, which correlated well with the subcellular distribution visualized by means of optical fluorescence microscopy.


  • McRae, R.; Lai, B.; Vogt, S., and Fahrni, C. J.: Correlative microXRF and optical immunofluorescence microscopy of adherent cells labeled with ultrasmall gold particles. J. Struct. Biol., 155, 22-29 (2006).

The following references provide helpful protocols for labeling using FluoroNanogold for fluorescence and electron microscopy:

* Alexa Fluor is a registered trademark of Invitrogen (Molecular Probes), Inc.

More information:

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FluoroNanogold In Situ Immunolabeling for Placental Protein Localization

John Robinson has published many papers describing applications of FluoroNanogold, and adds to these with a report in Placenta on its use for in situ immunolabeling in order to obtain detailed protein localization information within the amnion epithelium. Detailed information regarding the subcellular distribution of proteins within amnion epithelial cells is a goal of numerous placental biologists; however, morphological preservation is particularly challenging in this tissue, and therefore alternative immunolabeling and specimen preparation methods are desirable.

Following fixation with 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS; pH 7.4), amnion specimens were directly permeabilized with 0.1% saponin (0.1% saponin is a component of all buffers used in subsequent incubation and washing steps), blocked in 5% normal goat serum with 1% nonfat dry milk in PBS for 1 hour at room temperature, then incubated overnight with primary antibodies at 4°C. Antibodies were evaluated directed against COX-1, COX-2, microsomal PGES-1, microsomal PGES-2, cytosolic PGES, and adipose differentiation-related protein (ADRP). Specimens were then exposed sequentially to biotinylated goat anti-rabbit or goat anti-mouse antibodies, followed by Alexa Fluor® 594 FluoroNanogold-Streptavidin, with intervening washing steps. In control specimens, the primary antibody was substituted for an equivalent concentration of purified pre-immune rabbit IgG or isotype-specific mouse IgG. Portions were mounted onto standard microscope slides and visualized by conventional epifluorescence and confocal laser scanning microscopy. Remaining specimens were subjected to silver-based autometallography to evaluate whether the technique was also applicable to transmission electron microscopy.

For comparison, in parallel experiments, specimens were fixed in 4% PFA/PBS for 1 hour at room temperature, and membrane roll cryosections (~ µm) were prepared. Serial sections were stained with hematoxylin and eosin (H&E) to evaluate overall morphology, and selected specimens immunolabeled using the same antibodies and incubation conditions. Additionally, primary cultures of amnion cells were established and grown to confluence on glass coverslips for 5 days, fixed in 4% PFA/PBS for 1 hour at room temperature, and labeled using the conditions described for the in situ specimens. To induce COX-2 expression, some of the cells cultured in vitro were challenged for 4 hours with a cytokine (10 ng/ml of recombinant human interleukin-1b) prior to fixation. These specimens were visualized using epifluorescence and confocal microscopy.

This versatile technique for in situ immunolabeling in amnion is as technically permissible as traditional immunolabeling of cultured cells. When coupled with confocal laser scanning microscopy, it is similarly capable of providing detailed information regarding subcellular protein distribution. Using antibodies directed against sequential enzymes of the prostaglandin E biosynthesis cascade, this novel method was compared with immunofluorescent labeling, using amnion cells in primary culture and cryosections of reflected fetal membrane rolls. By several criteria, morphological variation was observed between the cells cultured in vitro and the tissue specimens. Immunostaining patterns were generally consistent between the cryosectioned specimens and those labeled in situ; however, better morphological preservation was found in the in situ stained specimens. Relative to the cryosectioned specimens, in situ immunostaining permitted improved sampling efficiency, and allowed regional variations in labeling to be observed in a more global context within the tissue. In situ immunolabeling therefore provides a useful addition or alternative to immunolabeling using membrane roll preparations.

[Correlative Alexa Fluor 594 FluoroNanogold Labeling (67k)]

Correlative fluorescence and electron microscopic labeling with Alexa Fluor 594-Streptavidin: Localization of caveolin-1a in ultrathin cryosection of human placenta; caveolin 1 alpha is primarily located to caveolae in placental endothelial cells. One-to-one correspondence is found between fluorescent spots (upper right) and caveola labeled with gold particles (lower right). Ultrathin cryosections, collected on formvar film-coated nickel EM grids, were incubated with chicken anti-human caveolin-1a IgY for 30 min at 37°C, then with biotinylated goat anti-chicken F(ab')2 (13 µg/mL, 30 minutes at 37°C), then with Alexa Fluor 594 FluoroNanogold-Streptavidin (1:50 dilution, 30 minutes at room temperature). Non-specific sites on cryosections were blocked with 1% milk - 5% fetal bovine serum-PBS for 30 minutes at room temperature (figure courtesy of T. Takizawa, Ohio State University, Columbus, OH).


  • Ackerman, W. E., IV; Hughes, L. H.; Robinson, J. M., and Kniss, D. A.: In situ immunolabeling allows for detailed localization of prostaglandin synthesizing enzymes within amnion epithelium. Placenta, 27, 919-923 (2006).

More information:

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Gold Labeling of RNA and Oligonucleotides...Revisited

A number of previous articles have addressed various aspects of oligonucleotide labeling, and since we are still frequently asked how to prepare Nanogold®-labeled oligonucleotides, it may be helpful to review the various approaches.

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.


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

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.


  • 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).

  • 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).

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).

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.


  • 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).

DNA Labeling

The best characterized approach to DNA labeling is to synthesize your oligonucleotide in the desired sequence with a suitable reactive group, then prepare Nanogold®-labeled DNA using one of our Nanogold labeling reagents. First, you will need to 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: you can then label these using Monomaleimido Nanogold or Mono-Sulfo-NHS-Nanogold respectively. DNA oligonucleotides may be prepared with these functional groups 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. Examples of this approach are shown below:

[Synthesis of DNA with modified phosphoramidites and labeling with Nanogold (106k)]

Two examples of the synthesis and labeling of chemically functionalized oligonucleotides, showing the use of modified phosphoramidites to introduce a 3'-thiol for labeling with Monomaleimido Nanogold (upper) and a 5'-amine for labeling with Mono-Sulfo-NHS-Nanogold (lower).

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).

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

Amino-modified oligonuleotides are mixed with reconstituted Mono-Sulfo-NHS-Nanogold at a pH between 7.5 and 8.2, incubated overnight at 4°C, then separated next day by gel filtration, an alternative chromatographic method, or by gel electrophoresis.

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).

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.

Plasmid Labeling

If you are working with a plasmid or other enzymatically-generated or naturally occurring oligonucleotide, you can't use modified phosphoramidites. What are the options in this case? There are several possibilities. These are discussed in detail in a recent newsletter article, and include 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. 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.

  • Use a photoreactive cross-linker to introduce a reactive site or a hapten into the completed plasmid. Good sources of cross-linking reagents with a wide variety of functionalities include Pierce, who provide a wide range of different types of reactivity, functionality and cleavability, and Molecular Biosciences. 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.

  • 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.


  • 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).

More information:

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Role of Sfi 1PCentrin Filaments in Yeast Revealed by Nanogold®

Nanogold®-Fab' fragments are among the smallest gold immunoprobes available. The 1.4 nm Nanogold particle is cross-linked directly to the conjugate Fab' fragment without an intervening layer of polymer or protein: therefore, these probes provide the highest resolution, and because the gold does not require stabilization by proteins, PEG or other macromolecules, the overall probe size is not significantly larger than unmodified Fab', allowing easiest penetration, easiest access to hindered antigens, and the highest possible labeling density. Nanogold conjugates can provide sufficient resolution to localize specific regions within a macromolecular complex, or to identify the orientation of a specific macromolecule relative to a known cellular structure. Li, Kilmartin and group used these advantages to help elucidate the structural role of Sfi 1pcentrin filaments in budding yeast spindle pole body duplication in their current and recent papers in the Journal of Cell Biology.

Centrins are calmodulin-like proteins, present in centrosomes and yeast spindle pole bodies (SPBs), and have essential functions in their duplication. The Saccharomyces cerevisiae centrin, Cdc31p, binds Sfi 1p on multiple conserved repeats. Both proteins localize to the SPB half-bridge, where the new SPB is assembled. The crystal structures of Sfi 1pcentrin complexes containing several repeats indicate that Sfi 1p is an alpha-helix with centrins wrapped around each repeat, and similar centrincentrin contacts between each repeat. Electron microscopy (EM) was used to investigate the structure of an Sfi 1pcentrin complex with 15 Sfi 1 repeats and 15 bound centrins. EM rotary shadowing of the 15-repeat Sfi 1pcentrin complex in 1 mM EGTA or 1 mM CaCl2 was used to determine its length; equal volumes of 50% glycerol and sample (20/~ µg/ml) in 500 mM NaCl with 20 mM Tris-C1, pH 8.0, and 1 mM DTT were sprayed onto freshly cleaved mica, vacuum dried for 2 h, and rotary shadowed with platinum at an angle of 6°. Combined with nanospray mass spectrometry measurements, this yielded an observed average length for the filaments of close to 60 nm, consistent with the arrangement of all the Sfi 1 repeats as a continuous alpha-helix. This suggests that the full complex containing 21 repeats could be 90 nm long: this is long enough to span the 60-nm half-bridge with the remainder localized in the central plaque, consistent with the staining of centrin spread along the mainly cytoplasmic side of the bridge.

Immunoelectron microscopy, using Sfi 1p tagged with GFP either at the N-terminus or the C-terminus by recombinant PCR methods, was used to localize the protein and, from the comparative positions of the stain, its orientation and the supramolecular organization of the filaments. The immunoEM protocol was developed specifically to detect transient SPB duplication intermediates which might be rarely present, even in synchronized cells. A preembedding staining method was used for greater sensitivity; however, a major problem with SPB antigens is that even short periods of formaldehyde fixation (1 minute for Spc42p) can abolish their reactivity with both polyclonal and monoclonal antibodies. With Spc42p, a number of different epitope tags were investigated, including single and multiple myc and hemagglutinin tags at the -NH2 and -COOH termini, but green fluorescent protein (GFP) was found to be far superior in retaining reactivity after formaldehyde fixation. During processing for immunoEM, the cells fractured open; this was advantageous because the need for detergent permeabilization was removed and the nuclear membrane, which was necessary for the proper identification of the half-bridge, satellite, and duplication plaque, could be preserved.

Cells were harvested by centrifugation, washed once with water, then fixed in 3.7% formaldehyde solution, in 0.1 M potassium phosphate, pH 6.5, for 20 minutes at 22°C. After three washes with 0.1 M potassium phosphate, pH 6.5, and one with 0.1 M phosphate citrate buffer, pH 5.8 (PC), cells were incubated with 10% v/v glusulase and 0.1 mg/ml zymolyase 20T in PC at 30°C for 1 hour. Samples were then washed once with PC, incubated with 50 mM glycine in PBS at 4°C for 5 minutes, washed twice with 0.5 ml PBS-BSA, and incubated with either affinity-purified rabbit anti-GFP antibody diluted 1:150 in PBS-BSA, 1% solution P (90 mg PMSF, 2 mg pepstatin in 5 ml absolute ethanol), 30100 µg/ml of purified 9E10 or 12CA5, or with affinity-purified rabbit anti-Tub4p diluted 1:3,000 for 1 hour at 22°C. After washing three times with PBS-BSA, samples were then incubated with a 1:50 dilution of Nanogold-labeled goat antirabbit Fab' or 1:20 dilution of goat antimouse Fab' in PBS-BSA, 1% solution P at 22°C for 1 hour. Samples were washed once with PBS-BSA and three times with PBS, then fixed with 2.5% glutaraldehyde in 40 mM potassium phosphate, pH 6.5, 0.5 mM MgCl2 for 2 hours at 22°C. The glutaraldehyde was removed by washing once with buffer, then incubating three times for 5 minutes each with 50 mM MES with 200 mM sucrose at pH 6.0 at 4°C. Silver enhancement was performed in the dark for 35 minutes at 22°C with NPG silver enhancement solution (reference below); the samples were then washed three times with cold 200 mM MES, pH 6.15, in the dark over 10 minutes, removed from the dark room and washed twice with 0.1 M sodium acetate, pH 6.1. Following postfixing with 2% osmium tetroxide, samples were processed for serial thin section EM. In thin-section EM, bridge length was measured as the distance between the edge of the central plaques to the edge of the second central plaque or satellite or duplication plaque. Half-bridge length was measured from the edge of the central plaque to the end of the electron-dense nuclear membrane or cytoplasmic outer layer. Half-bridge and bridge length between paired SPBs was measured from log phase cells, and other measurements were from alpha factorblocked or released cells.

ImmunoEM localization of the Sfi 1p N and C termini showed Sfi 1pcentrin filaments spanning the length of the half-bridge with the Sfi 1p N terminus at the SPB. This suggests a model for SPB duplication where the half-bridge doubles in length by association of the Sfi 1p C termini, thereby providing a new Sfi 1p N terminus to initiate SPB assembly. To determine whether the N and C termini of Sfi 1p are distant from each other, with the N terminus associated with the edge of the central plaque, labeling distribution for N and C termini were quantified by plotting as the presence or absence of silver deposition for 10-nm sectors along the SPBs and bridge (the labeling structure itself, before silver deposition - rabbit IgG with Fab' antirabbit-Nanogold - would span ~20 nm). The data showed a different distribution for the N- and C-terminal GFP: the N-terminal label is always close to the junction between the SPB and the proximal end of the half-bridge or bridge, and in paired SPBs shows two separate localization sites close to each SPB; in contrast, the N-terminal label is either close to the distal end of the halfbridge or close to the center of the bridge in paired SPBs. The N-terminal label also showed another class of labeling for single SPBs that appeared to have full bridges, either SPBs at a very early stage of duplication when daughter SPB components start to assemble, or satellite-bearing SPBs or paired SPBs where it was not possible to locate a satellite or second SPB in the serial sections. Staining with both labels was always cytoplasmic. These data indicate that the N terminus of Sfi 1p lies close to the edge of the central plaque and the filamentous Sfi 1pcentrin repeats span the length of the half-bridge, ending with the C terminus of Sfi 1p at the distal end of the half-bridge. During duplication, the doubling in the length of the bridge takes place by the end-to-end addition of another molecule of Sfi 1p by association of their C termini, and the N terminus of this second Sfi 1p molecule is associated with the edge of the central plaque of the daughter SPB.


  • Li, S.; Sandercock, A. M.; Conduit, P.; Robinson, C. V.; Williams, R. L., and Kilmartin, J. V.: Structural role of Sfi1p-centrin filaments in budding yeast spindle pole body duplication. J. Cell. Biol., 173, 867-877 (2006).

  • Kilmartin, J. V.: Sfi1p has conserved centrin-binding sites and an essential function in budding yeast spindle pole body duplication. J. Cell. Biol., 162, 867-877 (2003).

More detailed immunoelectron microscopy procedure:

Silver enhancement procedure:

More information:

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Ordering and Custom Synthesis: Some Reminders

If you have a question about an order, such as whether it has been received or shipped, you should contact our main office (nano@nanoprobes.com; telephone 1-877-447-6266 in North America, ++ (631) 205-9490 from elsewhere, fax (631) 203-9493) rather than technical support. Details of individual orders, shipping, and billing are not accessible to technical support personnel.

If you are looking for Nanogold® conjugate or reagent that is similar to our catalog items and the chemistry has already been established - such as a multiply functionalized Nanogold particle, or a Nanogold-labeled primary antibody - we can usually prepare such products as custom syntheses. Please contact technical support (tech@nanoprobes.com) if you have a specific custom synthesis request. If your request includes steps that we do not do regularly, such as labeling a different type of protein or biomolecule, we can often consider it, but may need to treat it as a short-term contract research project in which payment is required whether or not the synthesis is successful.

For your information, contact information is summarized below:

Question: Contact Telephone E-mail
Ordering, order status, shipping or payment Sales office 1-877-447-6266 or (631) 205-9490 nano@nanoprobes.com
Product availability or delivery time Sales office 1-877-447-6266 or (631) 205-9490 nano@nanoprobes.com
Technical question or custom synthesis Technical support 1-877-447-6266 or (631) 205-9492 tech@nanoprobes.com
Problem with product Technical support 1-877-447-6266 or (631) 205-9492 tech@nanoprobes.com
Business inquiry or general information General business office 1-877-447-6266 or (631) 205-9490 nano@nanoprobes.com

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Other Recent Publications

The reliability of Nanogold® staining was also demonstrated by Luo and co-workers in their recent investigation into the role of Schwann cell differentiation in perineural invasion of adenoid cystic and mucoepidermoid carcinomas of the salivary glands in the International Journal of Oral and Maxillofacial Surgery. Twenty cases of adenoid cystic carcinoma (ACC) and 18 cases of mucoepidermoid carcinoma (MEC), were examined for expression of the Schwann cell markers S100 protein and glial fibrillary acidic protein (GFAP) by immunohistochemical staining, and the relationship between S100 and GFAP expression and the occurrence of perineural invasion assessed. Ultrastructural localization of S100 and GFAP was examined by immunoelectron microscopy. Tumor sections obtained during operations, fixed immediately in ice-cold 4% paraformaldehyde / 0.05% glutaraldehyde (pH 7.4) were divided, cryoprotected with PBS containing 25% sucrose and 10% glycerol, and blocked with PBS containing 5% bovine serum albumin and 5% normal goat serum for 4 hours. These specimens were incubated overnight in primary antibody (rabbit anti-human S100, diluted 1:80, or mouse anti-human GFAP, diluted 1:50 in PBS - 1% bovine serum albumin - 1% normal goat serum), washed in PBS then incubated overnight with a 1:100 dilution of Nanogold goat anti-rabbit / mouse IgG, rinsed, postfixed in 2% glutaraldehyde in PBS for 45 min and enhanced in the dark with HQ Silver, rinsing before and after with de-ionized water. Immunolabelled sections were fixed with 0.5% osmium tetroxide in 0.1 M phosphate buffer for 1 hour, dehydrated in graded ethanol followed by propylene oxide, and flat-embedded in Epon 812. By light microscopy, no S100/GFAP immunoreactivity was found in MEC sections, but most ACCs showed strong intensity staining for S100/GFAP. 3-4 Sections showing S100/GFAP immunoreactivity were selected from each ACC specimen and mounted. Ultrathin sections were cut, mounted on mesh grids, and counterstained with uranyl acetate and lead citrate. Co-expression of S100 and muscle actin in the same type of ACC cells was found by double fluorescence immunostain. Perineural invasion was found in 11 ACCs (55%) and 0 MECs (0%). S100 and GFAP were expressed inmost of the ACCs but none of the MECs; a significant difference was observed in the rate of perineural invasion and expression of S100 and GFAP between ACC and MEC (P < 0.001). Expression of S100 and GFAP correlated with perineural invasion in salivary malignancy (P < 0.001), and the ultrastructures of S100- and GFAP-positive cells were consistent with the characteristics of myoepithelial cells. These results indicate that Schwann cell differentiation correlates with perineural invasion in salivary malignancy, and occurs in modified myoepithelial cells of ACC.


  • Luo, X.-L.; Sun, M.-Y.; Lu, C.-T., and Zhou, Z.-H.: The role of Schwann cell differentiation in perineural invasion of adenoid cystic and mucoepidermoid carcinoma of the salivary glands. Int. J. Oral Maxillofac. Surg., 35, 733-739 (2006).

Chad Mirkin and group were back in the Journal of the American Chemical Society this month with a novel method for the DNA-induced separation of gold nanoparticles of different sizes in aqueous media, utilizing the inherent chemical recognition properties of DNA and the cooperative binding properties of DNA-functionalized gold nanoparticles. Melting temperatures (Tms) of aggregates formed from nanoparticles interconnected by duplex DNA were found to be dependent upon particle size: this effect is proposed to derive from larger contact areas between the larger particles and consequent increased cooperativity, leading to higher Tms. Separation was achieved by taking two aliquots of a mixture of particles of varying size, and functionalizing each with complementary DNA. The aliquots were mixed at a temperature above the Tm for aggregates formed from the smaller particles, but below the Tm for aggregates formed from the larger particles. The resulting aggregates consist almost exclusively of the larger particles and were easily separated from the smaller suspended particles by sedimentation and centrifugation. The method was used to separate binary mixtures of 30/60, 15/60, and 40/80 nm gold particles functionalized with complementary decanucleotides, and also a ternary mixture of 30, 50 and 80 nm gold particles.


We have frequently discussed the fluorescence quenching properties of gold nanoparticles, in previous articles on FluoroNanogold, Molecular Beacons and methods for combined fluorescent and larger gold labeling, as well as our recent report on the combination of Nanogold® immunolabeling with the ReAsH tag. In the current Angewandte Chemie International Edition in English, Sapsford, Berti and Medintz comprehensively review recent developments in the field of FRET, with applications including the structural elucidation of biological molecules and their interactions, in vitro assays, in vivo monitoring in cellular research, nucleic acid analysis, signal transduction, light harvesting and metallic nanomaterials. In addition to the phenomena mentioned above, other novel combinations of quenchers and fluorophores such as nanocrystals, nanoparticles, polymers, and genetically encoded proteins are discussed. This review gives a critical overview of the major classes of fluorophores that may act as donor, acceptor, or both in a FRET configuration, focussing on the benefits and limitations of these materials and their combinations, and available methods for their bioconjugation.


  • Sapsford, K. E.; Berti, L., and Medintz, I. L.: Materials for Fluorescence Resonance Energy Transfer Analysis: Beyond Traditional Donor-Acceptor Combinations. Angew. Chem. Int. Ed. Engl., 45, 4562-4589 (2006).

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