N A N O P R O B E S     E - N E W S

Vol. 10, No. 8          August 31, 2009

Updated: August 31, 2009

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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NTA-Ni(II)-Nanogold® Helps Assign 3-D Structure of Cav3.1

NTA-Ni(II)-Nanogold® is a unique labeling reagent in which the targeting agent is not an antibody or a protein but a nickel (II) nitrilotriacetic acid (NTA) chelate, which targets polyhistidine (His) tags. His tags may be readily engineered into most recombinant proteins, and this makes NTA-Ni(II)-Nanogold a potential universal secondary reagent for use with engineered or recombinant proteins and peptide probes, as well as an ideal choice for localizing overexpressed His-tagged proteins. NTA-Ni(II)-Nanogold® is much smaller than Nanogold®-Fab', and consequently can access targets that antibodies cannot, and can often label protein subunits without changing the structure of the assembled protein. Because of the proximity of the NTA-Ni(II) chelate to the gold - only about 1.5 nm - it also provides much higher labeling resolution, and it can be used for macromolecular and even molecular structural methods, such as Cryoelectron microscopy and, as demonstrated this month by Walsh and co-workers, high-resolution electron microscopy with single-particle analysis.

Features of NTA-Ni(II)-Nanogold include:

  • Higher labeling resolution. The NTA-Ni(II) chelate is much smaller than an antibody or protein: when it is bound, the gold is within about 1.5 nm of the target. This makes NTA-Ni(II)-Nanogold ideal for localizing subunits in protein complexes or specific sites in macromolecular assemblies at molecular resolution.

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

  • Smaller size means less ultrastructural perturbation: it is ideal for labeling proteins in their natural configuration.

  • Binding constants for Ni(II)-NTA are very high due to the combination of the chelate effect of multiple histidine binding, and target binding of multiple Ni(II)-NTA groups on each gold particle. Dissociation constants are estimated to be between 10-7 to 10-13 M-1, comparable to antibodies for many applications.

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

[Ni-NTA-Nanogold structure and STEM (48k)]

Left: Structure of Ni-NTA-Nanogold, showing the binding of the incorporated metal chelate to a His-tagged protein. Inset shows the resolution, expressed as the distance from the center of the Nanogold particle to the His tag: note that this is significantly shorter than the equivalent distance with antibodies. 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).

Calcium entry through voltage-gated calcium channels affects a wide range of physiological processes. Neuronal excitability, muscle excitation-contraction coupling, and secretion are all influenced by calcium flux, and therefore the mechanisms by which calcium is distributed are important targets for study. Using single particle analysis methods, Walsh and group have determined the first three-dimensional structure, at 23 Å resolution, of one member of the low voltage-activated voltage-gated calcium channel family, recombinant Cav3.1, a T-type channel.

Recombinant Cav3.1 was expressed in Sf9 cells: Cav3.1 containing a C-terminal c-Myc and decahistidine (His10) tag was subcloned into the baculovirus vector pFastBac-HTa. Sf9 insect cells were cultured in TNM-FH medium supplemented with 10% (v/v) fetal bovine serum and split when confluent. For protein expression, 7080% confluent Sf9 cells were infected with recombinant baculoviruses at a multiplicity of infection of 12, then incubated at 27°C for 72 hours. Virus-infected Sf9 cells expressing the Cav3.1 were centrifuged at 1000 x g for 5 minutes, resuspended in 10 mL of 20 mM Tris, pH 8.0, with 150 mM NaCl and 1 mM DTT plus protease inhibitor, then lysed by sonication for 3 x 30 seconds. After centrifugation at 1000 x g for 5 minutes to remove large particulate matter, the Sf9 cell membranes were pelleted by centrifugation at 50,000 x g for 1 hour. The pellet was resuspended in 10 mL of 20 mM Tris, pH 8.0, containing 150 mM NaCl, 20 mM imidazole, 1 mM DTT and 1% (w/v) CHAPS with protease inhibitors, incubated for 1 hour on a rolling platform at 4°C. Unsolubilized material was removed by centrifugation at 20,000 x g for 30 minutes at 4°C, the resulting supernatant was diluted 1:1 with 20 mM Tris buffer, pH 8.0, with 150 mM NaCl, 20 mM imidazole, and 1 mM DTT, and the His-tagged protein recovered chromatographically using a 1 mL Ni-NTA column pre-equilibrated with 20 mM Tris buffer, pH 8.0, with 150 mM NaCl, 20 mM imidazole, 1 mM DTT and 0.5% (w/v) CHAPS. The protein solution was eluted by the addition of 1 column volume of equilibration buffer containing 350 mM imidazole.

For the gold labeling, excess Ni-NTA-Nanogold was added to the purified Cav3.1 and incubated for 3 hours. Aliquots were then stained with uranyl acetate. Both unlabeled and labeled Cav3.1 were examined using the same electron microscopic procedure, and analyzed using the same initial start model.

Unlabeled and labeled Cav3.1 samples were examined using standard negative staining electron microscopic techniques with 2% (w/v) uranyl acetate. Images were recorded between a 0.651.05 µm defocus range at 100 kV, at a calibrated magnification of x 47,800. Micrographs were scanned at 3.6 Å/pixel at the specimen level. The contrast transfer function (CTF) was determined for each image using CTFIT (part of the EMAN software); images were corrected for phase effects only. The resolution of the final three-dimensional volumes were assessed using established procedures: each data set was divided into two, with volumes calculated for the two subsets, and the resolution was estimated by the Fourier shell correlation (FSC) coefficient with the resolution limit taken to be where the FSC value fell below 0.5. All models were generated using the UCSF Chimera program.

The three-dimensional reconstruction of recombinant Cav3.1 was generated using the common line projection matching methods employed in EMAN software, with no symmetry applied (C1). A wide variety of starting models were tested including Gaussian blobs, transmembrane domain of KV1.2 (PDB entry 2R9R, edited), and the full KV1.2 crystal structure (2R9R) filtered to an appropriate resolution. In all reconstructions, the principal structural features were consistent, indicating the fidelity of the final three-dimensional reconstruction. For the unlabeled protein, approximately 11,500 particles were selected interactively using the graphical interface boxer (EMAN image processing software) into a box of 64 x 64 pixels (230 x 230 Å); approximately 4,500 gold-labeled Cav3.1 particles were selected, and a good sampling of different orientations of labeled Cav3.1 was observed.

Particles were band-pass-filtered and centered, and a preliminary three-dimensional model was calculated from a set of unbiased reference-free class averages showing different orientations of the complex (refine2d.py program under EMAN). The three-dimensional model was iteratively refined at each step using a projection matching routine, whereby projections with uniformly distributed orientations of the preliminary three-dimensional model were used as references for classification of the raw data set, with the class averages from this step used to construct a new three-dimensional model. Refinement was carried out with an angular coverage of 7° generating 388 classes with between 10 and 48 particles per class. Final reconstruction included 86% of the selected images. Convergence (stabilization of the three-dimensional structure), was monitored by examining the FSC of the three-dimensional models generated from each iteration.

Cav3.1 has dimensions of ~115 x 85 x 95 Å. It is composed of two distinct segments. The cytoplasmic densities form a vestibule below the transmembrane domain with the C terminus, which was unambiguously identified by the presence of a His tag and attached gold label: this was ~65 Å long, and curls around the base of the structure. The cytoplasmic assembly has a large exposed surface area, which may serve as a signaling hub, where the C terminus acts as a "fishing rod" to bind regulatory proteins. A three-dimensional structure was also determined at a resolution of 25 Å for the monomeric form of the cardiac L-type voltage-gated calcium (high voltage-activated) channel with accessory proteins beta and alpha2delta bound to the ion channel polypeptide Cav1.2. Comparison with the skeletal muscle isoform finds a good match particularly with respect to the conformation, size, and shape of the domain identified as that formed by alpha2. Modeling of the Cav3.1 structure (analogous to Cav1.2 at these resolutions) into the heteromeric L-type voltage-gated calcium channel complex volume reveals multiple interaction sites for beta-Cav1.2 binding and for the first time identifies the size and organization of the alpha2delta polypeptides.


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

[Ni-NTA-5 nm Gold structure, and TEM View (141k)]

Top: Structure of NTA-Ni(II)-5 nm Gold, showing the binding of the incorporated metal chelate to a His-tagged protein; distance from the gold particle surface to the His tag is estimated to be 1.5 nm. Above: Transmission electron micrograph of 5 nm NTA Gold: average diameter 5.11±0.84nm.

Features and advantages:

  • High visibility: 5 nm gold is clearly visualized at TEM resolution without silver or gold enhancement. Labeling is simplified and more monodisperse: you can use this probe for multiple labeling studies in conjunction with gold particles of different sizes, or with silver or gold-enhanced Nanogold.

  • Precise labeling resolution: when the probe is bound, the gold is much closer to its target: about 1.5 nm. This makes NTA-Ni(II)-Nanogold ideal for localizing molecular sites in protein complexes or other macromolecular assemblies.

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

  • Strong binding: binding constants for Ni(II)-NTA-5 nm Gold are very high due to the combination of the chelate effect of multiple histidine binding, and binding of multiple Ni(II)-NTA functionalities on each gold. Dissociation constants are estimated to be between 10-7 to 10-13 M-1, comparable to antibodies.

  • High penetration: 5 nm Ni-NTA-Gold is smaller than an unlabeled primary antibody, penetrates better, and accesses more sterically restricted interior sites. It may be used with stronger fixation or less permeabilization, enabling labeling with better ultrastructural preservation.

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

Applications of Ni-NTA-Gold probes include:

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

Product details:

Product name Catalog number Gold particle size Unit size Price (USD)
Ni-NTA-Nanogold 1.8 nm 2080 30 nmol $342.00
5 nm Ni-NTA-Gold 5 nm 2082 1 or 2 mL at OD(516nm) = 2.0 $382.00


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


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Should I Use Nanogold®-Fab' or IgG?

Here's a question that has come up twice in the last couple of weeks: what is the difference between Nanogold®-Fab' (such as catalog number 2004, Nanogold-Fab' Goat anti-Rabbit IgG) and Nanogold-IgG conjugates (such as catalog number 2003, Nanogold-IgG Goat anti-Rabbit IgG), and which one should I use?

The difference refers to the antibody or fragment to which the Nanogold label is conjugated. Because Nanogold is covalently linked using different chemical reactions, it may be conjugated to specific chemical sites in a variety of antibody fragments instead of whole IgG molecules, including Fab' fragments, Fab fragments, and even ScFv fragments. Of these, we offer a range of secondary antibody conjugates comprising Nanogold attached to either whole IgG antibody molecules, or Fab' fragments. These conjugates have different properties, and these may make them more or less appropriate for different applications.

[Nanogold-IgG and Fab' (78k)]

Structure and preparation of Nanogold-IgG and Nanogold-Fab' conjugates. Note that while Nanogold-IgG retains the Fc fragment and has two binding sites, Nanogold-Fab' does not contain the Fc fragment and has only one binding site: however, Nanogold-Fab' is considerably smaller, and in fact is less than half the size of an unlabeled IgG.

Nanogold-Fab' fragments are smaller, can label more densely, and penetrate better into cells and tissue sections, and access antigens that are more hindered. Since they also do not contain Fc, they therefore cannot be targeted by Fc-specific tertiary probes, or interact with specimens through Fc-specific mechanisms. This may be better for double labeling, since one mechanism for cross-reactivity or non-specific interaction is removed.

Nanogold-IgG, since it includes the whole IgG, can provide stronger binding since it has two antigen binding sites. Since it contains Fc, it can also be targeted by Fc-specific tertiary probes.

Both reagents will provide effective immunoelectron microscopic staining with silver enhancement; your choice should be dictated by which of the sets of properties above fits better with your experiment and with the system in which the probes will be used.

The key features and differences between these two Nanogold probes are summarized below:

Reagent: Nanogold-IgG Nanogold-Fab'
Penetration: High penetration: will penetrate into cells and tissues better than many colloidal gold probes. Highest penetration, up to 40 microns into cells and tissues: Takizawa and Robinson report significantly better penetration with Nanogold-Fab' than with Nanogold-IgG.
Labeling density: Relatively high, but may not be quantitative. Very high. 1 : 1 ratio of Nanogold : Fab' means one Nanogold per binding site and produces quantitative labeling if all antigens are accessible.
Antigen combining regions: Two - can give a chelate effect with stronger binding. One - gives nearest to quantitative labeling.
Fc region: Yes No
  • Labeling surface antigens where binding strength is at a premium.
  • Labeling protocols where a Fc-specific tertiary probe may be required.
  • Immunogold labeling in delicate or less strongly fixed specimens.
  • Binding studies in solution.
  • Labeling interior antigens where penetration is at a premium.
  • Quantitative labeling.
  • Situations where Fc interactions might lead to background or non-specific interactions, such as multiple labeling studies.

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


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Inexpensive Conductimetric Biochips with EnzMetTM

EnzMetTM (Enzyme Metallography) is a new method, developed by Nanoprobes, in which a targeted enzyme-labeled probe is used to deposit metal from solution. EnzMetTM has proven to be a very clean and sensitive method for in situ hybridization (ISH) and immunohistochemistry (IHC), as well as for electron microscopy (EM). However, its applications extend beyond the optical. Wolfgang Fritzsche, Robert Möller and co-workers have applied it successfully to the development of an electrical detection method for biochips. Because multiple independent electrical contacts can be fabricated and identified on a microscopic scale, electrical detection offers the potential for highly multiplexed target detection in a robust, miniaturized and highly portable format. Previous studies demonstrated that EnzMetTM avoids the background found when using silver-enhanced gold to form the contacts, yielding improved signal-to-noise.

[Enzyme metallography electrical detection (49k)]

Enzyme metallography as an electrical detection method. left: the enzyme metallographic process. right: design of electrical detection system: target oligonucleotide is deposited between electrodes, detected with enzymatic probe which is then "developed" with enzyme metallography to produce a conductive connection.

Having demonstrated proof of principle, the group then pursued ways to fabricate this type of biochip affordably, and in the current issue of Biosensors and Bioelectronics, report an inexpensive microfluidic format that may make this method rapid and cost-effective for many DNA detection applications. By enclosing the chip within an inexpensive microfluidic flow cell, the operation of the detection system was simplified and the operator intervention minimized.

The microarray measurement platform is a microstructured glass chip with screen printed electrodes and 42 measurement points (electrode gaps). The capture DNA molecules are immobilized in the gap between the electrodes. The DNA chips were chemically modified with (3-glycidyloxypropyl)-trimethoxysilane (GOPS) in order to bind amino-modified single stranded (ss) capture-DNA molecules. First, the chips were cleaned by sonication for 5 minutes each with acetone, ethanol, and water. After drying with nitrogen, the chips were modified in a 10mM GOPS solution in dry toluene for 7 hours at 70°C, then finally washed 2 X 5 minutes each with toluene, ethanol, and water. The deposited capture DNA was modified at the 5' or 3' end with a C6-Aminolink, which was then attached to the epoxy (introduced by GOPS) modified surface. After the specific binding of biotinylated target molecules, a streptavidin-horseradishperoxidase-polymer was bound in the electrode gap. EnzMetTM-induced deposition of silver nanoparticles leads to bridging the electrode gap by a conductive silver layer: the increase of the conductivity over the electrode gap can be measured by a straightforward DC measurement, managed using an attached PC; a heating foil and thermocouple are also incorporated into the polycarbonate flow cell cover.

Slightly differently to the conventional (non-microfluidic) system, the best specificity for the hybridization of the biotin-labeled target-DNA sequences was achieved in 0.5 X SSC buffer and 0.1% SDS hybridization buffer at 42°C. Hybridization was performed in the microfluidic device using a tubing pump with a planetary drive at a flow rate of 600 µL/min in 3 s intervals for 3 minutes. Subsequent washing in 0.2 X SSC for 1 minutes (in continuous flow) removed unbound DNA to avoid false positive signals. The streptavidin-horseradishperoxidase-polymer was diluted 1:1000 in PBS buffer and 0.05% Tween-20. This buffer was also used for the washing step, which was applied after a 3 minute incubation step. Enzyme binding was also carried out at the same pump parameters as those used for the hybridization. After binding of the streptavidin-enzyme conjugate, the EnzMetTM solution was mixed and introduced to the chip surface. Reaction was stopped after 3 minutes to avoid non-specific silver deposition. Before enzyme-induced silver deposition, the flow cell was rinsed with deionized water to remove any chloride from the PBS buffer.

An important factor for a bioanalytical tool is the total analysis time. In this system, the active movement of the different solutions by interval pumping means that binding processes are no longer solely diffusion limited, which should lead to faster reactions and higher binding efficiencies. To investigate this, different hybridization times were tested using a 500 nM concentration of biotin modified target DNA. A weak signal was already detectable for 30 second hybridization time. The signal intensity was found to increase up to 5 minutes, but with longer hybridization times, up to 15 minutes, the signal intensity approaches in saturation. Binding was confirmed by optical measurements. In all, a complete hybridization experiment could be completed within one hour using the flow cell, but required 4 hours in its absence. The detection limit of 50 pM biotinylated target DNA was comparable with other methods. Overall, this approach provides a robust, fast and cost-effective method for NA detection which promises to be readily applicable to use in the field.


  • Schüler, T.; Kretschmer, R.; Jessing, S.; Urban, M.; Fritzsche, W.; Möller, R., and Popp, J.: A disposable and cost efficient microfluidic device for the rapid chip-based electrical detection of DNA. Biosens. Bioelectron., 15-21 (2009).

  • Möller, R.; Schüler, T.; Günther, S.; Carlsohn, M. R.; Munder, T., and Fritzsche, W.: Electrical DNA-chip-based identification of different species of the genus Kitasatospora. Appl. Microbiol. Biotechnol., 77, 11811188 (2008).

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Labeling for EM and LM with Silver-Enhanced Nanogold® and DAB

Immunoenzymatic labeling is a viable method for both light and electron microscopic labeling, although the contrast of the deposited DAB is sometimes less than ideal for EM. However, because DAB generally produces a relatively diffuse and continuous stain of medium to low contrast, it is readily differentiated from the particulate staining pattern produced by gold or silver-enhanced gold: therefore, the combination of enzymatic labeling with silver-enhanced Nanogold® immunolabeling provides a method for double labeling which has been used to generate significant results for a number of publications. Constantinos Paspalas and colleagues compare the two methods at the light and electron microscopic level and add to their extensive publication record in this field with an entry in the current Cerebral Cortex.

[Silver-enhanced Nanogold and enzymatic double labeling (186k)]

Left: Sequential double labeling, showing (b) labeling of first target with gold and silver enhancement, followed by (c) labeling of second target using peroxidase developed with DAB. Right: Double labeling with silver-enhanced Nanogold and immunoperoxidase. Subcellular distribution of m4R immunoreactivity in striatal cholinergic neurons of control rats (A) or rats treated with oxotremorine (B). Cholinergic neurons were identified by immunoreactivity for ChAT, detected by the immunoperoxidase method: m4R reactivity was detected by Nanogold pre-embedding labeling with HQ silver enhancement. The ChAT immunoreaction product was visible throughout the cytoplasm of the perikarya. (A) The neuron immunopositive for ChAT and m4R has a large volume of cytoplasm, typical of a striatal interneuron, with immunoparticles at the external surface of the ER (arrows), Golgi apparatus (G), and external nuclear membrane ( flat arrow). Very few are associated with the plasma membrane (arrowheads). (B) After treatment with oxotremorine, most immunoparticles are clearly associated with endoplasmic reticulum lamina (arrows). Scale bars, 1 mm.

Regulator of G protein signaling 4 (RGS4) regulates intracellular signaling via G proteins and is markedly reduced in the prefrontal cortex (PFC) of patients with schizophrenia. Therefore, characterizing the expression of RGS4 within individual neuronal compartments is important to understanding its actions on individual G protein - coupled receptors (GPCRs), and ultimately the nature of schizophrenia and related disorders. The authors address this challenge by presenting an ultrastructural reference map of RGS4 protein in macaque PFC, based on immunogold electron microscopic analysis. In order to better identify specific targets, the authors used a tertiary labeling protocol in order to deposit 'clusters,' or groups of gold labels, at each target site, and to verify the distribution at the light and EM level, compared enzymatic staining with silver-enhanced Nanogold.

3 adult rhesus macaques were used for this study: the primates were deeply anesthetized prior to transcardial perfusion of artificial cerebrospinal fluid, followed by 4% paraformaldehyde/0.05 - 0.1% glutaraldehyde in 100 mM phosphate-buffered saline. Brains were sectioned coronally at 60 µm, cryoprotected, and stored at -80°C. Sections of the dorsolateral PFC (Walkers area) were processed for RGS4 immunocytochemistry. In order to facilitate penetration of immunoreagents, all sections went through 3 freeze-thaw cycles in liquid nitrogen. Nonspecific reactivity was blocked with 10% normal donkey serum (NDS) and 2% bovine serum albumin (BSA) in 50 mM Tris-buffered saline (TBS). Immunoaffinity purified RGS4 antibody (IgY) was raised in chicken against amino acids 127-205 of the RGS4 protein C-terminus: this antibody recognizes human and rodent RGS4 based on sequence homology.

Immunocytochemistry for light microscopy was conducted with both DAB and silver-enhanced Nanogold. For enzymatic staining, sections were incubated for 36 hours in 1 : 1000 anti-RGS4 in Tris-buffered saline (TBS) with 0.05% Triton X-100, 2% normal serum (NDS), and 1% bovine serum albumin (BSA), then for a further 3 hours in 1 : 500 biotinylated F(ab')2. A 1 : 100 dilution of avidin-biotin-peroxidase complexes (Vector Laboratories) was then applied for one hour. For Nanogold-silver staining, a 1 : 100 Nanogold-labeled anti-biotin IgG was applied for 2 hours. RGS4 was then visualized either with diaminobenzidine as an enzyme chromogen, or by silver enhancement of Nanogold after glutaraldehyde fixation using HQ Silver. For immunoelectron microscopy, sections were placed in 1 : 2000 anti-RGS4 for 48 hours, followed by 1 : 800 biotinylated F(ab')2, both diluted in TBS with 2% NDS, 0.1% acetylated BSA-C (Aurion), and 0.01% Tween-20 (gold buffer). Next, a 1 : 200 dilution of Nanogold-IgG anti-biotin in gold buffer was applied, followed by silver enhancement (autometallography) to visualize the Nanogold particles. As there are multiple gold probes per primary antibody, this 3-layer immunoprocedure was expected to produce multiparticle aggregates, or 'particle clusters.' Omission of the anti-RGS4 or substitution with normal chicken serum abolished all reactivity. When bridging biotinylated antibodies were excluded, both Nanogold and peroxidase signals were eliminated. Similarly, peroxidase labeling was abolished when blocking the biotinylated probes with avidin/biotin. To control for self-nucleation of the metallographic developer, Nanogold was omitted and the sections processed for silver enhancement for 15 minutes at room temperature. In RGS4 labeling, enhancement time never exceeded 8 min at 4°C. All controls were evaluated under the electron microscope. At the light microscopic level, a faint diffuse precipitate remained in control sections treated with diaminobenzidine, but was not detectable ultrastructurally. Matching sections were processed in parallel for gold immunocytochemistry against antigens with known distribution patterns to test the validity of nuclear labeling for RGS4.

For electron microscopy, layers I-III and IV-VI of PFC were sampled for resectioning and analysis under the transmission electron microscope at 80 kV. Twenty plastic blocks of each brain were examined using only the 4th to 12th surface-most sections of each block (i.e., 200-600 nm): signal sharply declined with depth from the tissue surface, possibly due to the larger size of the IgY primary antibody. RGS4 structures were digitally captured at X 25,000 to X 160,000 magnification and individual panels adjusted for brightness and contrast. Quantitative assessments were performed on series of low magnification micrographs of supragranular PFC, each covering a field of 30 µm2 captured from the 4th and the 12th thin section to avoid recounting a single structure.

Examination of the micrographs revealed that at the soma, all labeling was asynaptic and affiliated with subsurface cistern microdomains of pyramidal neurons. Most immunoreactivity was associated with the nucleus. RGS4 levels were particularly high along proximal apical dendrites, and markedly decreased with distance from the soma; clustered Nanogold labels were observed at the bifurcation into second-order branches. In distal dendrites and in spines, the protein was found flanking or directly facing the postsynaptic density of symmetric and asymmetric synapses. Axons also expressed RGS4, and the density and distribution of pre- and postsynaptic labeling was correlated with the axon ultrastructure and the type of established synapses. These data indicate that RGS4 is strategically positioned to regulate not only postsynaptic but also presynaptic signaling in response to synaptic and nonsynaptic GPCR activation. This gives it broad but highly selective influence on multiple aspects of PFC cellular physiology.


  • Paspalas, C. D.; Selemon, L. D., and Arnsten, A. F.: Mapping the regulator of G protein signaling 4 (RGS4): presynaptic and postsynaptic substrates for neuroregulation in prefrontal cortex. Cereb. Cortex., 19, 2145-2155 (2009).

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Nanoprobes Wins New SBIR Grant

Nanoprobes has received a new Small Business Innovation (SBIR) Research grant from the National Institute of Biomedical Imaging and Bioengineering (one of the National Institutes of Health (NIH)). This 6-month, Phase 1 study will support research on covalently linked gold labels and probes for targeting proteins at the electron microscopic level, and will enable us to continue to develop our core technologies of gold nanoparticle functionalization and bioconjugate chemistry. The work will be directed by Dr. Vishwas Joshi.

We also welcome new Research Assistant Ping Lin to our company. Ms. Lin will assist with the development of gold as a labeling reagent and as a biomedical imaging reagent.

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Other Recent Publications

Ottenheijm and group provide a nifty demonstration of high-resolution Nanogold® labeling for electron microscopy in their studies of Nemaline myopathy (NM) described in their paper in Human Molecular Genetics: the used phalloidin-biotin followed by Nanogold-Streptavidin to label actin in a mouse nebulin knockout model. Small muscle bundles were dissected and skinned overnight. Skinned bundles were fixed in 3.7% paraformaldehyde, and labeled overnight with phalloidin-biotin, then incubated with Nanogold®-Streptavidin and silver enhanced using HQ Silver and examined by electron microscopy. For the determination of thin filament density, fibers were cross-sectioned (75 nm thick sections stained with uranyl acetate and lead citrate) and then longitudinally for sarcomere length measurements. Combined with immunofluorescence confocal microscopy, the electron microscopy studies indicated that average thin filament length is reduced from ~1.3 mm in control muscle to ~0.75 mm in NM-NEB muscle. This study is the first to show a distinct genotype-functional phenotype correlation in patients with NM due to a nebulin mutation, and provides evidence that dysregulated thin filament length contributes to muscle weakness in NM patients with nebulin mutations. A striking similarity between the contractile and structural phenotypes of nebulin-deficient mouse muscle and human NM-NEB muscle was observed, indicating that the nebulin knockout model is well suited for studying the functional basis of muscle weakness in NM and therapeutic development in humans.


  • Ottenheijm, C. A.; Witt, C. C.; Stienen, G. J.; Labeit, S.; Beggs, A. H., and Granzier, H.: Thin filament length dysregulation contributes to muscle weakness in nemaline myopathy patients with nebulin deficiency. Hum. Mol. Genet., 18, 2359-2369 (2009).

Meanwhile, applications of our negative stains, NanoVan and Nano-W keep coming. The latest paper by Odegard and co-workers in the Journal of Virology describes the use of Nano-W negative staining to study viral infection. The process by which nonenveloped viruses cross cell membranes during host cell entry is currently poorly defined, and the authors used correlated in vivo and in vitro studies to elucidate the mechanism of Flock House virus (FHV) entry and membrane penetration. For electron microscopic studies, FHV particles were stained with 2% Nano-W (methylamine tungstate), and the samples viewed in a transmission electron microscope at 100 keV. Low endocytic pH was found to be required for FHV infection: exposure to acidic pH promotes FHV-mediated disruption of model membranes (liposomes), and particles exposed to low pH in vitro exhibit increased hydrophobicity. In addition, FHV particles perturbed by heating displayed a marked increase in liposome disruption, indicating that membrane-active regions of the capsid are exposed or released under these conditions. Evidence was also found that autoproteolytic cleavage, to generate the lipophilic gamma peptide (4.4 kDa), is required for membrane penetration. Mutant, cleavage-defective particles failed to mediate liposome lysis regardless of pH or heat treatment, indicating that these particles are not able to expose or release the requisite membrane-active regions of the capsid - the gamma peptides. An updated model for FHV entry is proposed, in which the virus enters the host cell by endocytosis, low pH within the endocytic pathway triggers the irreversible exposure or release of gamma peptides from the virus particle, and the exposed/released gamma peptides disrupt the endosomal membrane, facilitating translocation of viral RNA into the cytoplasm.


  • Odegard, A. L.; Kwan, M. H.; Walukiewicz, H. E.; Banerjee, M.; Schneemann, A., and Johnson, J. E.: Low endocytic pH and capsid protein autocleavage are critical components of Flock House virus cell entry. J. Virol., 83, 8628-8637 (2009).

Gold nanoparticles can facilitate electron transfer, and therefore can be used to amplify redox reactions. One application of this is the use of gold nanoparticle-modified electrodes for the detection of analytes that can undergo an electrochemical reaction; an example was presented by Teresa Luczak in a recent paper in Electrochemica Acta. The author used mercaptopropionic acid (MPA), gold nanoparticles (Au-NPs) and cystamine (CA) modified gold bare electrodes as voltammetric sensors for simultaneous detection of epinephrine (EP), ascorbic (AA) and uric (UA) acids. Colloidal gold nanoparticles some 10 - 15 nm in diameter were prepared using a conventional citrate reduction of tetrachloroaurate, and an activated (polished) "2D" gold electrode was immersed in the colloidal gold solution for 24 hours at 4°C. Both the bare gold ("2D") and the nanoparticle-modified ("3D") electrodes were then coated with MPA Modification of the electrode surface by self-assembled layers (SAMs) by immersion in a 5mM ethanolic solution of MPA for 10 hours. The presence of the SAM was found to improve the reactivity of the gold electrode for EP oxidation remarkably. A linear relationship between the epinephrine concentration and the current response was obtained in the range of 0.1700 µM with a detection limit of 0.042 µM for gold electrodes, and in the range of 0.1800 µM with the detection limit of 0.040 µM for the electrodes incorporating gold nanoparticles. The results have shown that the overlapping voltammetric responses of epinephrine, ascorbic and uric acids are well resolved at modified electrodes. The modified SAMs electrodes show high selectivity, sensitivity, reproducibility and stability.


  • Luczak, T.: Comparison of electrochemical oxidation of epinephrine in the presence of interfering ascorbic and uric acids on gold electrodes modified with S-functionalized compounds and gold nanoparticles Electrochim. Acta, 54, 5863-5870 (2009).

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