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N A N O P R O B E S     E - N E W S

Vol. 8, No. 8          August 22, 2007

Updated: August 22, 2007

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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Combined Fluorescent and Larger Gold Labeling

Our combined fluorescent and gold immunoprobe, FluoroNanogold, in which both a fluorescent label and the 1.4 nm Nanogold® particle are conjugated to a Fab' fragment, have enabled several different combinations of labeling techniques, including correlative fluorescent and gold labeling, fluorescent and scanning electron microscopic labeling, and combined fluorescence and X-ray fluorescent labeling. However, because gold nanoparticles are effective fluorescence quenchers, this approach is limited to the 1.4 nm Nanogold: combined fluorescence and larger gold probes do not work because the gold particles quench the fluorescence. Previous approaches to combined larger gold and fluorescent labeling have relied on fluorescently labeled secondary antibodies with gold-labeled tertiaries, which require additional steps and reduce resolution at the EM level.

At Microscopy and Microanalysis 2007, we introduced a new type of probe that allows combined fluorescent and 5 or 10 nm gold labeling in a single step. This new probe contains and enzyme and a gold particle conjugated separately to an IgG molecule: once the probe has bound to its target, treatment with a fluorescent or fluorogenic substrate results in selective deposition of the substrate at the target site to give fluorescent labeling; subsequently, the gold particles may be localized by electron microscopy. The fluorophores are deposited between 10 and 100 nm from the enzyme: this is lower than the resolution limit for fluorescence and hence there is no reduction in fluorescence resolution, but far enough from the gold particles that bright, usable fluorescence is maintained. Dual-labeled probes comprising horseradish peroxidase-labeled antibodies or streptavidin conjugated to 1.4 nm Nanogold, 5 nm or 10 nm colloidal gold were prepared, and used with fluorescent tyramide HRP substrates for fluorescent labeling. The structure and mechanism of these probes, and an example of the results obtained with a combined peroxidase and 5 nm gold probe, are shown below.

[Combined enzymatic and gold probes and labeling (83k)]

Combined enzymatic and gold probes. (a): Schematic showing structure and action of combined enzymatic and gold probes depositing a fluorescent or fluorogenic substrate to give combined fluorescent and larger gold labeling. (b) and (c): Labeling of polar tube proteins in B. algerae microsporida in cultured RK-13 cells, stained using anti-PTP-80 primary antibody followed by combined HRP and 5 nm gold-labeled secondary antibody. (b): Fluorescence after development with Alexa Fluor*® 488 tyramide for 15 minutes (40X objective); (c) TEM without silver enhancement or enzyme metallographic development, showing gold particles (arrows). (d) Immunoblot detection of serial dilutions of biotinylated IgG using (left) HRP and Nanogoldlabeled streptavidin, and (right) HRP-streptavidin, developed with enzyme metallography: note the increase in sensitivity of two orders of magnitude with the combined enzymatic and gold probe.

The new probes were evaluated in Microsporida, parasitic organisms that are important intracellular opportunistic pathogens in AIDS and other immune compromised patients. Microsporida are responsible for chronic diarrhea, malabsorption syndromes, myositis, and disseminating infections in all tissues of the body: all form a diagnostic spore containing a coiled polar filament surrounding the sporoplasm, consisting of a nucleus or paired abutted nuclei (diplokaryon) and associated cytoplasmic organelles. This polar filament everts to form a tubule through which the spore contents travel to infect a host cell upon activation. Cultured RK-13 cells infected with Brachiola algerae microsporida were grown in plastic cell culture chamber slides. After immunofixing and blocking, the infected cells were incubated with 1:100 anti- B. algerae PTP80 primary antibody for 30 minutes at 32°C, followed by either a 1 : 200 dilution of Nanogold and HRP-labeled IgG, or a 1 : 50 dilution of 5 or 10 nm gold and HRP-labeled IgG secondary for 30 minutes at 32°C. A 10 or 15 minute treatment with Alexa Fluor*® 488 tyramide revealed the polar tubes (PTs) clearly by fluorescence microscopy at similar resolution to FluoroNanogold, even using probes containing 5 or 10 nm gold. Fluorescence labeling was still clear even after silver enhancement of Nanogold. Transmission electron microscopy showed specific, dense PT labeling with 5 and 10 nm gold. This is the first demonstration of correlative 5 or 10 nm gold and fluorescent labeling using a single probe. Silver enhancement produced dense labeling of PTs, observable by both LM and EM.

These probes may have other important benefits both to biomedical research and for public health. Enzyme metallography produced extremely intense labeling of the polar tubes, readily observed by both LM and EM. In conjunction with fluorescent enzyme substrates, this approach enables correlative fluorescent, brightfield LM and EM studies on the same structures. With enzyme metallography, the intense labeling of the 150 nm diameter PTs revealed their structure much more clearly than previous approaches. Previously, diagnostic identification of microsporidial infection has required electron microscopy; the prevalence of these infections in less developed regions of the world, where they are a major public health issue, means that this is usually not practical. These new probes may enable the diagnostics allows the identification of microsporida with low power brightfield LM optics, thus providing a much more robust and inexpensive diagnostic capability for use in remote or less developed regions; this has many potential advantages, including more effective therapy selection, increased longevity and better quality of life for HIV pateints, more accurate allocation of therapeutics to less developed countries. In addition, on immunodot blots, the new probes showed significantly greater detection sensitivity than enzyme metallographic detection or Nanogold with silver enhancement, perhaps due to mutual reinforcement of the enzymatic redox activity by the adjacent gold nanoparticle: this indicates potential applications in detection procedures such as Western and Southern blotting where radiolabeling and chemiluminescence are currently the only options with sufficient sensitivity.


  • Powell, R.; Joshi, V.; Takvorian, P.; Cali, A., and Hainfeld, J.: Correlative Enzymatic and Gold Probes for Light and Electron Microscopy. Microsc. Microanal., 13, (Suppl. 2: Proceedings) (Proceedings of Microscopy and Microanalysis 2006); Marko, M.; Scott, J.-H.; Vicenzi, E.; DeKanich, S.; Frafjord, J.; Kotula, P.; McKernan, S., and Shields, J. (Eds.),; Cambridge University Press, New York, NY; p. 244CD (2007).

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

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Labeling Fc or Hinge IgG Sites with Nanogold®

Although our first choice for labeling IgG with Nanogold® is to use Monomaleimido Nanogold to label at a hinge thiol, this approach may not be ideal for all systems. Several alternative methods are available, with advantages and disadvantages. Two which may be conducted readily using Nanogold reagents are oxidizing a sugar residue in the glycosylated Fc region of the IgG and labeling with Monoamino Nanogold, and labeling at an amino- site (either an N-terminal amine or a lysine residue) using Mono-Sulfo-NHS-Nanogold. These three methods are discussed and compared below.

[Synthetic strategies for IgG labeling with Nanogold (152k)]

Synthetic strategies for labeling IgG with Nanogold. (Top) Labeling at a hinge thiol with Monomaleimido Nanogold gives the greatest site-specificity; however, labeling at an Fc sugar residue (middle) ensures that labeling is remote from the antigen binding regions, while using Mono-Sulfo-NHS-Nanogold to label at the N-terminal position or at a lysine residue (bottom) can provide a more straightforward synthesis, and works on most types of IgG under similar conditions.

Hinge thiol labeling with Monomaleimido Nanogold

This method provides the greatest degree of control over labeling. Use it for:

  • High resolution electron microscopic labeling.
  • Antigen quantitation and other quantitative labeling applications.
  • Where you have sufficient antibody to repeat the labeling reaction if labeling is low first time.
The thiol group that is used for labeling is obtained by the reduction of a hinge disulfide, which is usually conducted using a reducing agent such as dithiothreitol (DTT), mercaptoethylamine hydrochloride (MEA) or mercaptoethanol. The susceptibility of the hinge thiols to reduction varies between different IgG types and species, but the optimum concentration for reducing hinge disulfides while leaving disulfides elsewhere intact is usually between 10 mM and 50 mM: reduction is usually carried out for 60 to 90 minutes at slightly elevated temperature (37°C). The antibody is then separated from the reducing agent using gel filtration over a desalting column such as GH25 desalting gel (contact Millipore): this is required since the thiol-based reducing agent will react with maleimides. The reduced IgG is then mixed with Monomaleimido Nanogold at pH 6.5 at room temperature, agitated gently for one hour, then incubated overnight at 4°C to ensure that the reaction is complete. The labeled IgG is then separated from excess Nanogold by gel filtration using a Superose-12 or similar column.

Although we have not extensively tested this alternative, the labeling procedure may be simplified by using a non-thiol based reducing agent, such as sodium borohydride (NaBH4) or sodium cyanoborohydride (NaBH3CN), which do not react with maleimide. This removes the requirement to separate the reducing agent; however, allowing it to quench before adding the Monomaleimido Nanogold is recommended, as these may still possess some reactivity towards components of the Nanogold label.


  • Most site-specific labeling.
  • Conjugates labeled at the hinge region give highest electron microscope labeling resolution.
  • Attachment site is positioned away from both the antigen combining and Fc regions, allowing both to participate in binding reactions (for example, binding a tertiary probe).


  • Moderately complex labeling procedure: requires gel filtration to separate thiol-based reducing agents.
  • Hinge thiols in IgGs from different species vary in their susceptibility to reduction. Some trial and error may be necessary to identify the optimum conditions for hinge thiol reduction.

Fc labeling using periodate oxidation and Monoamino Nanogold

Labeling at the Fc region is somewhat more challenging than the conventional hinge thiol labeling approach because the Fc region has less well-defined functional groups. One unique group that is present in the Fc region of antibodies is carbohydrates, or sugar residues. You can use mild oxidation with sodium periodate to convert these to reactive aldehydes; the aldehydes may then be labeled with Monoamino Nanogold, followed by reduction with cyanoborohydride to reduce the resulting Schiff base to a secondary amine. This procedure is actually the same as that recommended in our Application Note on RNA labeling. This approach is recommended when:

  • You have a limited amount of antibody, require labeling away from the antigen binding region, but do not know the optimum conditions for hinge disulfide reduction.
  • You wish to leave the hinge region for subsequent reaction.

Alternatively, it may be possible to convert the aldehydes to carboxylic acids. If so, the carboxylic acid may be converted to a reactive ester using either 1-Ethyl-3-[3-dimethylaminopropyl] carbodiimide Hydrochloride (EDC) with Sulfo-N-hydroxysuccinimide or N,N-carbonyldiimidazole in DMF: the activated ester is then reacted with Monoamino Nanogold.

You can also cross-link to aldehydes directly using a carbonyl-reactive hydrazido- maleimide cross-linkers; a variety are available. Succinimidyl 6-(3-[2-pyridyldithio]-propionamido) hydrazide (SPDP hydrazide) introduces a disulfide, which is readily reduced to a sulfhydryl and labeled with Monomaleimido Nanogold. Others introduce maleimides, which may then be reacted with mercaptoethylamine hydrochloride to convert them to amines, and labeled with Mono-Sulfo-NHS-Nanogold. You can find suitable cross-linkers from the list of heterobifunctional cross-linkers from Molecular Biosciences, or the cross-linker selection guide from Pierce.


  • Nanogold label is located well away from the antigen combining region, so maximum immunoreactivity is preserved.
  • Hinge thiol is preserved for maximum structural integrity.
  • Procedure is more straightforward than hinge thiol labeling, since gel filtration is not required before Nanogold addition.
  • A variety of cross-linking options are available.


  • Labeling results may be variable.
  • Because the gold is positioned away from the binding region, resolution in the electron microscope is lower than it would be with hinge thiol labeling.
  • Oxidation and reduction reactions can produce multiple labeling sites, making labeling more difficult to control than hinge thiol labeling.

N-terminal or lysine residue amine labeling using Mono-Sulfo-NHS-Nanogold

The simplest method for IgG labeling, but also the least specific, is to react directly with Mono-Sulfo-NHS-Nanogold: this reagent will label any accessible amine, whether the N-terminal amine, or a lysine residue or other amino-functionalized amino acid residue or modification. Because IgG molecules usually contain multiple amine sites, this approach engenders a high level of confidence in labeling with little or no optimization. Because no modification is needed prior to conjugation, native antibody structure is preserved best with this method, and it is therefore recommended if other approaches result in changes to the immunoreactivity or structure of the IgG or a loss of stability after conjugation. This approach is useful if:

  • You have very little antibody or a very limited budget, and cannot afford to repeat the labeling reaction.
  • You need high ratios of gold to antibody: because most IgG molecules contain multiple amines, the likelihood of conjugating more than one Nanogold per antibody is higher than with hinge thiol labeling.
  • You have limited preparation time or your access to chromatography facilities is restricted.
  • If other methods result in loss of stability or antibody reactivity, or structure or MW changes.

The IgG is dissolved in a non-amine-containing buffer, such as phosphate-buffered saline or Tris-NaOH, at pH 7.5 to 8.2. Higher pH produces faster labeling, and may improve labeling efficiency, although it will also accelerate the competing hydrolysis of the NHS ester. The Mono-Sulfo-NHS-Nanogold is then reconstituted in water and mixed with the protein, agitated gently for 45 minutes to one hour, then incubated overnight at 4°C to ensure completion of the reaction. Next day, the labeled IgG is separated from excess Nanogold by gel filtration using a Superose-12 or similar column.


  • Simplest procedure for IgG labeling, and requires least preparation and equipment use.
  • Reaction most likely to work first time.
  • High ratios of Nanogold to IgG are possible.
  • Native IgG structure is best preserved, and may result in higher conjugate stability.


  • Because labeling can occur in any region of the IgG, this approach provides least control over the conjugation site and ratio of Nanogold to IgG.
  • Labeling may occur close to antigen binding region, possibly compromising immunoreactivity.
  • Conjugates labeled in this manner may provide lower resolution than others in macromolecular electron microscopic localizations.

More information:

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Undecagold with Cryoelectron Microscopy: Probing Myosin Interactions

In a recent report in the Journal of Molecular Biology, Lowey and colleagues demonstrated the advantages of undecagold, the smallest commercially available cluster label, for labeling myosin components that were then exchanged into myosin subfragment-1 (S1). Using cryoelectron microscopy with image analysis and averaging, the authors were able to detect the gold signal in assembled myosin at a higher resolution than that possible with direct TEM observation of gold labeling.

The src-homology 3 (SH3) domain in class II myosins is a distinct beta-barrel structure whose function remains unknown. The authors used a combination of electron cryomicroscopy, light-scattering, fluorescence and kinetic analysis to support the hypothesis that the SH3 domain facilitates the binding of the N-terminal extension of the essential light chain isoform (ELC-1) to actin. The 41 residue extension contains four conserved lysine residues, followed by a repeating sequence of seven proline and alanine residues; it is widely believed that the highly charged region interacts with actin, while the proline / alanine - rich sequence forms a rigid tether bridging the ~9 nm distance between the myosin lever arm and the thin filament.

Chicken pectoralis muscle myosin, and actin prepared from chicken acetone powder were used for the studies. Skeletal muscle S1 was prepared by chymotryptic digestion of chicken muscle myosin, then fractionated into S1(A1) and S1(A2) isoforms. Wild-type A1 was prepared from a chicken fast skeletal muscle cDNA clone. To localize the N terminus of ELC in the actomyosin complex, a cysteine residue was engineered into myosin using site-directed mutagenesis to change Ser45 to Cys, and a double Cys mutant (-C3/C45) was prepared by cloning C45 into the Cys vector; in the Cys mutants, the endogenous Cys at position 178 was changed to Ala (C178A). These were then reacted with a four- to five- fold molar excess of maleimido undecagold; the cluster is composed of a dense, central 11-atom gold core (~0.8 nm in diameter) stabilized and solubilized by a shell of organic groups (total diameter ~2 nm). Labeling of the -3Cys mutant was performed in 50 mM NaPi (pH 6.6), 10 mM NaCl at 4°C overnight. Free reducing agent was removed before labeling. Unbound gold cluster was removed by means of DEAE-Sephacel chromatography. The extent of labeling was determined spectrophotometrically, using the undecagold extinction coefficient of 47,000 M-1cm-1 at 420 nm. Protein concentration was determined with the Bradford (Pierce) colorimetric assay. S1(A1) in 30 mM KCl, 20 mM TrisHCl (pH 7.5) with 3 mM NaN3 was reacted with a twofold molar excess of Monomaleimido Undecagold for 3 hours on ice. Under these conditions, the K+-ATPase was reduced relative to a control, indicating labeling of the reactive thiol (SH1).

To prepare frozen-hydrated specimens for electron cryomicroscopy, F-actin (0.0250.03 mg/mL) was applied to glow-discharged 400-mesh copper grids coated with holey carbon film in 20 mM NaCl, 5 mM NaPi with 1 mM MgCl2 and 2 mM NaN3, pH 7.0. After a one minute incubation in a humid chamber, the grids were rinsed twice with myosin buffer without the myosin sample (10mM NaCl, 10mM imidazole, 1mM MgCl2 and 1mM DTT with 2 mM NaN3, pH 7.0,). Myosin sample, diluted to ~0.5 mg/ml or 2 mg/ml was then applied to the grid for 30 seonds, then replaced by an additional drop of sample (30 seconds). After excess liquid was blotted, the grids were plunged into liquid ethane cooled by liquid N2. Low-dose images were recorded using a CM12 electron microscope equipped with a LaB6 filament and cryoholder at a nominal magnification of 60,000x (120 keV), at ~1.5 µm defocus (electron dose ~10e--2). Micrographs were digitized with a pixel size of 0.27 nm; one data set containing S1(A1) with gold-labeled A1, one set containing S1(A1) with gold-labeled SH1, one data set of S1(A2) and three data sets from different preparations of unlabeled S1(A1) were obtained.

Localization of the gold labels was accomplished using difference maps, calculated by subtracting the S1 maps from the gold-labeled maps to give a total of four difference maps per label (SH1 and A1 labels). Maps were aligned to each other using the CoAn algorithm before subtraction, then for each gold label, the difference maps were averaged and the voxel-wise variance calculated. This was used to test the significance of features in the (averaged) difference maps using Student's t-test (confidence level 99.5%). Difference maps between the helical reconstructions did not show any statistically significant peak for the A1-labeled map; however, for the SH1 label, a single significant peak was found, and for the IHRSR maps, one significant peak was identified for each of the labels (SH1-labeled, and A1-labeled). For the SH1-labeled maps, the helical reconstruction difference peak corresponded to the highest peak in all IHRSR difference maps in the average; this was not the case for the A1-labeled maps, indicating disorder or partial occupancy. Multi-reference refinement was then applied to the A1-labeled data set using S1(A1) and S1(A1) with the A1-labeled peak from the averaged difference maps as references. The IHRSR reconstruction using that subset of the data was then used for difference mapping, which showed an enhanced peak in the same location: this was now the highest peak in each of the nonaveraged difference maps.

[Undecagold structure and labeling sites on S1-Actin (77k)]

Left: Structure of Monomaleimido Undecagold, showing the diameter of the gold core (tab) and ligand sphere (green). Right: Sites of undecagold labeling (top) difference peak on actin decorated with S1 containing undecagold-labeled A1 (undecagold labeled at Cys3), and (bottom) location of the difference peak attributed to the gold label on the reactive cysteine (SH1) of the heavy chain.

The electron cryomicroscopy of S1-bound actin filaments, together with computer-based docking of the skeletal S1 crystal structure into 3D reconstructions, showed a well-defined peak for the gold cluster near the SH3 domain. Given that SH3 domains are known to bind proline-rich ligands, this supports the hypothesis that the N-terminal extension of ELC interacts with actin and modulates myosin kinetics by binding to the SH3 domain during the ATPase cycle.


  • Lowey, S.; Saraswat, LD.; Liu, H.; Volkmann, N., and Hanein, D.: Evidence for an Interaction between the SH3 Domain and the N-terminal Extension of the Essential Light Chain in Class II Myosins. J. Mol. Biol., 371, 902-913 (2007).

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NTA-Ni(II)-Nanogold® Used to Solve Cryo-EM Structure of TOR-KOG1

In their recent paper in Molecular Cell, Adami and group provide yet another demonstration of the power of NTA-Ni(II)-Nanogold® for localizing functional components of protein complexes at the macromolecular level, in this case the TOR-KOG1 from yeast cells. NTA-Ni(II)-Nanogold is targeted by nitrilotriacetic acid (NTA) nickel (II), a small metal chelate that binds highly selectively to His (polyhistidine) tags, instead of an antibody or protein: this provides it with higher resolution than any antibody or protein probe, positioning the gold label only 2.5 nm from the target site. His tags may be readily engineered into most expressed proteins, and therefore NTA-Ni(II)-Nanogold is a potential universal secondary reagent which can be used to gold-label any synthetic or expressed protein and peptide probes.

TOR is a member of the PI3-kinase-like protein kinases (PIKK): it is a large molecular weight protein (281 kDa) which functions as a central controller of cell growth by integrating signals from hormones, growth factors, and nutrients. It is the target of rapamycin, an antifungal agent used clinically as an immunosuppressant which also has promising potential as an anticancer drug. TOR is a large Ser/Thr protein kinase that is highly conserved in yeasts and higher eukaryotes. It assembles into two distinct multiprotein complexes: TORC1 and TORC2. One defining feature of TORC1 is the interaction of TOR with KOG1 (Raptor in mammals): it is also sensitive to a rapamycin-FKBP12 complex.

Gold labeling was performed by incubating TOR-KOG1 complexes, incorporating a His10 tag at the C-terminus of KOG1, with the 1.8 nm NTA-Ni(II)-Nanogold. Before labeling, the TOR-KOG1 complex was dialyzed to remove any remaining elution buffer from the purification of the His-tagged protein. Conjugates were formed by incubation with a 10 molar excess of NTA-Ni(II)-Nanogold for 30 minutes at 4°C. These were observed in the electron microscope after negative staining with 1% uranyl acetate. 339 particles appearing to have some putative gold density were extracted and refined with EMAN, using the unlabeled TOR-KOG1 complex as a reference. In some averages, a gold density was identified that was not present in the reference projection used for classification and alignment. The location of the gold on the 3D reconstruction was inferred by the orientation of the volume projection used as reference for alignment of each class. Since negative staining can produce a dark background around averages, only those averages containing a black spot within the actual density of the protein and which were not present in the reference projection (corroborated by difference mapping) were considered as possible positives in the class averages. Several 2D averages complied with these requirements; all were found to correspond to several different orientations of the 3D reconstruction, but the density maximum was detected in the same area, confirming the localization of the Nanogold label.

CryoEM of the TOR-KOG1 complex was performed after vitrification of the sample by plunging into liquid ethane. Samples were observed at 200 kV; images were recorded at 50,000 and different defocus on film and digitized with a Zeiss scanner, step 7 µm. Particles were extracted and averaged to a final 2.8 Å/pixel. Due to the small amount and low concentration of the purified material, only a few hundreds of single molecules were collected. Manipulation in EMAN was used to obtain reference-free averages of the cryoEM data. The 25 Å resolution structures of endogenous budding yeast TOR1 and a TOR-KOG1 complex reconstructed in three dimensions, using electron microscopy. TOR was found to feature distinctive N-terminal HEAT repeats that form a curved tubular domain, which associates with the C-terminal WD40 repeat domain of KOG1.

[NTA-Ni(II)-Nanogold structure, and labeling of the KOG1-TOR complex (58k)]

Left: Structure of NTA-Ni(II)-Nanogold, showing interaction with a His-tagged protein, and (upper right) resolution, or distance from target His tag to gold particle. Center: Structure of NTA-Ni(II)-Nanogold-labeled His-tagged KOG1-TOR in which the C terminus of KOG1 was tagged with a His10. Reference projections of the reconstruction were used to classify and average the data. Right: Knob protein from adenovirus cloned with 6x-His tag, labeled with Ni-NTA-Nanogold, column purified from excess gold, and viewed in the scanning transmission electron microscope (STEM) unstained (Full width approximately 245 nm).

The results show that TOR1 is organized into a bulky C-terminal region containing the helical/kinase, FAT and FATC domains, and an extended tubular region comprising the N-terminal HEAT repeats, similar to other PIKK-family proteins. The C terminus of KOG1, which comprises a seven-bladed propeller, interacts with the N terminus of TOR1, while the N-terminal RNC domain binds the TOR C terminus. This arrangement of the complex places the RNC domain of KOG1, which interacts with the TOS motifs of TORC1 substrates, in close proximity to the kinase domain of TOR. KOG1/Raptor is known to strongly stimulate TORC1 phosphorylation of S6K1 and 4E-BP1 by recruiting substrate to the TOR catalytic site. The structural and biochemical data presented in this report, which link the TOS-binding site of KOG1/Raptor with the kinase domain of TOR, are consistent with this substrate recruitment mechanism, confirming that the N terminus of KOG1 likely functions to bring substrates into the vicinity of the catalytic region. This architectural model also explains the sensitivity of the TOR-KOG1 complex to rapamycin.


  • Adami, A.; Garcia-Alvarez, B.; Arias-Palomo, E.; Barford, D., and Llorca, O.: Structure of TOR and its complex with KOG1. Mol. Cell., 27 509-516 (2007).

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Enzyme Metallography, and Other News

Check our News page for press releases and other news stories about Nanoprobes; from this page, you can also view archived press releases and news stories since we began issuing press releases in 1994. For current information, such as days when we will be closed, changes to our contact information, or issues that affect our ability to serve or respond to our customers, please check our current news and information page.

Currently, you can check our press release on the recent review on the development of metallographic methods for in situhybridization. The development of metallographic methods and their application to determination of HER2 gene amplification in breast cancer, including Nanogold® with silver enhancement, Nanogold with gold enhancement (GOLDFISH), and finally enzyme metallography, are reviewed in detail. Nanoprobes recently signed a deal with Ventana Medical Systems, Incorporated, for commercial development and use of this reagent in automated slide staining instruments. As a result, the first commercial product has now been introduced in Europe; it is called SISH (Silver In Situ Hybridization). Introduction of SISH in the United States is pending FDA approval. In addition, Nanoprobes will shortly introduce a commercial EnzMetTM formulation optimized for research applications and non-automated staining.

[Enzyme Metallography: schematic and comparison with DAB (74k)]

Enzyme metallography (EnzMet): (left) how it works. Comparison between conventional DAB detection with H&E counterstain (center) and EnzMet with nuclear fast red counterstain (right) detection of HER2 gene copies in paraffin-embedded infiltrating ductal carcinoma of the breast (micrographs courtesy of Raymond R. Tubbs, Cleveland Clinic Foundation).

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Other Recent Publications

Adami and co-workers were not the only group to report new results with NTA-Ni(II)-Nanogold®. Collins and group, who have previously published several papers describing the use of this reagent to characterize lipoprotein complexes, presented another result in the Journal of bacteriology in their studies of type IV pili, surface-exposed retractable fibers which play a key role in the pathogenesis of Neisseria meningitidis and other gram-negative pathogens. PilG, an integral inner membrane protein and component of the type IV pilus biogenesis system which is related by sequence to the extensive GspF family of secretory proteins involved in type II secretion processes, was overexpressed and purified from Escherichia coli membranes by detergent extraction and metal ion affinity chromatography. Analysis by perfluoro-octanoic acid polyacrylamide gel electrophoresis indicated that PilG formed dimers and tetramers: a three-dimensional (3-D) electron microscopy structure of the PilG multimer was then determined using single-particle averaging applied to samples visualized by negative stain cryoelectron microscopy. Symmetry analysis supported a tetrameric structure for the PilG multimer, and also revealed an asymmetric bilobed structure approximately 125 Å in length and 80 Å in width. The larger lobe within this structure was identified as the N terminus by location of NTA-Ni(II)-Nanogold® labeling to an N-terminal polyhistidine tag. The authors propose that the smaller lobe corresponds to the periplasmic domain of the protein, with the narrower "waist" region being the transmembrane section. This is the first report of a 3-D structure of a member of the GspF family, and suggests that there is a physical basis for the role of this protein in linking cytoplasmic and periplasmic protein components of the type II secretion and type IV pilus biogenesis systems.


  • Collins, R. F.; Saleem, M., and Derrick, J. P.: Purification and Three-Dimensional Electron Microscopy Structure of the Neisseria meningitidis Type IV Pilus Biogenesis Protein PilG. J Bacteriol., 189, 6389-6396 (2007).

Solodukhin and colleagues, meanwhile, were adding to the new results reported with our vanadium-based negative staining reagent, NanoVan. This reagent combines a light stain, which allows easy visualization of smaller gold labels such as Nanogold®, with a very fine grain structure that allows high structural resolution. In their article in Cellular Signalling, they prepared two-dimensional crystals of protein kinase C delta (PKCdelta) and of its regulatory domain (RDdelta) on lipid monolayers. 2D crystals of PKCdelta and RDdelta were grown on monolayers composed of dioleoyl-phosphatidylcholine (DOPC): dioleoyl-phosphatidylserine (DOPS): diolein (DO) (45:50:5, molar ratio). PKCdelta (48 nM) or RDdelta (5.4 nM) in 17 µl of 20 mM 3-(N-morpholino)-propanesulfonic acid, pH 7.8 was overlayed with 12 µL of the same buffer and then 1.0 µL of freshly mixed lipids (2.0 mg/mL in chloroform:hexane, 1:1) and incubated for 7 hours in darkness at 4°C. Membranes were picked up on carbon-coated lacey 300 mesh copper electron microscopy grids and stained with 1% Nanovan for 30 seconds, washed with distilled water, and dried for 1 hour. The crystal preparations were then analyzed by electron microscopy at tilt angles varying from ?50° to +55°. Although the crystals exhibit pseudo-3-fold symmetry, analysis of difference phase residuals indicated that there is only one way to align the crystals for merging so the data were processed in plane group P1. Three-dimensional reconstructions generated for several two-dimensional crystals of PKCdelta and RDdelta both showed good agreement, and were consistent with membrane attachment via a single C1 subdomain, a small surface contact by one or two loops from the C2 domain, and, in intact PKCdelta, a small appendage from the catalytic domain, probably V5. The technique of two-dimensional crystallography with three-dimensional reconstruction appears well suited to examination of additional PKC isozymes, as well as the analysis of the enzymes bound to substrates and other proteins.


  • Solodukhin, A. S.; Kretsinger, R. H., and Sando, J. J.: Initial three-dimensional reconstructions of protein kinase C delta from two-dimensional crystals on lipid monolayers. Cell. Signal., 19, 2035-2045 (2007).

A novel light scattering method for identification of different cell types based on differential biomarker expression was reported by Yu and colleagues in Nano Letters. Gold nanorod molecular probes (GNrMPs) were designed and fabricated for multiplex identification of cell surface markers in HBECs: nanorods with different aspect ratios are distinguished by spectral changes in scattered light. Gold nanorods with aspect ratios of 1.5, 2.8, and 4.5 were prepared by a wet-chemistry, seed-mediated growth method with an initial CTAB coating: the nanorods were activated by partial replacement of the CTAB with 11-Mercaptoundecanoic acid (MUDA), which was then used for activation and cross-linking to antibodies against CD24, CD44, and CD49f. The probes were used to probe cells directly, using dark field microscopy integrated with a spectral imager for simultaneous detection of the three surface markers. The immunophenotype composition of these cell lines, an indicator of their metastasis potential, was assessed using the GNrMPs. This technique, which may allow multiplexed detection of up to 15 markers, has the potential to become an important tool for diagnosis and prognosis of breast and other cancers.


  • Yu, C.; Nakshatri, H., and Irudayaraj, J.: Identity Profiling of Cell Surface Markers by Multiplex Gold Nanorod Probes. Nano Lett.,/cite> 7, 2300-2306 (2007).

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