N A N O P R O B E S     E - N E W S

Vol. 10, No. 5          May 31, 2009

Updated: May 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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Nanogold® for Electron Cryotomography

Nanogold® labeling reagents are a unique core technology of Nanoprobes. They allow the attachment of gold labels to specific sites on biological molecules and probes with the same precision as fluorescent labels. Compared with colloidal gold, these gold cluster labels have several important advantages:

  • Site-specific attachment allows labeling at a site that does not perturb native reactivity.
  • Nanogold labels are stabilized by a layer of small organic ligands, and do not require additional macromolecules for stabilization. Hence conjugates are smaller than those with equivalent sized colloidal gold, penetrate into cells and tissues better, and access restricted antigens better.
  • The ligand-encapsulated Nanogold surface is uncharged, hydrophilic and does not bind non-specifically: hence non-specific binding and aggregation are eliminated.
  • High labeling resolution and more quantitative labeling than colloidal gold, due to small probe size.

The small size and high penetration of Nanogold labels makes them ideal for imaging the 3-dimensional distribution of proteins or other targets in cells or tissues by electron tomography; the only issue has been that their small size can make them difficult to detect by conventional bright-field electron tomography, particularly in thicker sections. However, Björkman and co-workers, who have previously demonstrated the advantages of Nanogold for this application, confirm in their new paper in Microscopy and Microanalysis that, by using electron cryotomography (ECT), they can visualize Nanogold labeling even in 250 µm ice sections.

FcRn is a receptor that transfers maternal IgG across epithelial cell barriers to mammalian offspring to passively immunize the newborn against environmental antigens. It also serves as a protection receptor to rescue IgG in the blood from a default degradative pathway. In newborn rats, FcRn transfers IgG from milk to blood by apical-to-basolateral transcytosis across intestinal epithelial cells. This is facilitated by the pH difference between the apical (pH 6.06.5) and basolateral (pH 7.4) sides of intestinal epithelial cells: FcRn binds IgG at pH 6.06.5, but releases at higher pH values (7.4 and above), hence transport is unidirectional. While crystal structures of FcRn alone, and FcRn bound to Fc revealed the molecular details of the pH-dependent interaction between FcRn and IgG, but did show directly how membrane-bound FcRn interacts with and transports IgG inside intracellular vesicles. To investigate, the authors coupled FcRn to synthetic liposomes and incubated them with Nanogold-labeled Fc fragments, to imitate the interaction between membrane-bound FcRn and IgG, then used ECT to obtain 3D structural information on the membrane-associated FcRn-Fc complexes. The direct visualization of 1.4 nm gold clusters by ECT suggests that it can be a useful single molecular marker to map the distribution of receptor-ligand complexes on membranes.

The interaction was studied using synthetic liposomes prepared with 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidophenyl) butyramide] (MBP-PE), dried and then hydrated with 20 mM Hepes pH 6.5, 0.15M NaCl. A mini-extruder with a 0.2 µm membrane was used to prepare liposomes. A soluble version of rat FcRn containing an introduced free cysteine (the K19C FcRn mutant) was purified from the supernatants of transfected CHO cells, then incubated in 20mM DTT, desalted using a Spin 6 desalting column, then incubated overnight at room temperature with the MPB-PE liposomes in 20 mM Hepes pH 6.5, 150 mM NaCl. This resulted in covalent coupling of the single free cysteine in the K19C FcRn mutant to the MPB-PE lipids through a thioether reaction. FcRn-coupled liposomes were concentrated using a Centriplus 100 kDa cutoff centrifuge filter, then exchanged into 20 mM Hepes pH 6.0. Liposomes with diameters between 50 and 200 nm were estimated to contain 701,000 receptors per liposome, calculated by dividing the liposome surface area (785,00012,000,000 Å2) by 11,000 Å2, the approximate surface area covered by a glycosylated FcRn protein.

Several types of metal clusters were considered for labeling Fc. However, the authors were unable to observe FcRn binding by Fc proteins labeled with a 3 nm thiol-reactive monolayer-protected gold cluster; and Fc proteins labeled with ultrasmall (~1 nm) or larger colloidal gold clusters formed aggregates that did not bind detectably to FcRn. By contrast, Fc proteins labeled with 1.4 nm Monomaleimido Nanogold retained pH-dependent binding to FcRn, and showed no detectable aggregation as monitored by gel filtration chromatography. Purified rat Fc was labeled with Monomaleimido Nanogold according to usual protocols, producing Nanogold-labeled Fc that was separated from both unlabeled Fc and free Nanogold by gel filtration chromatography, then further purified by FcRn affinity chromatography. This procedure guaranteed that Nanogold-Fc preparations contained no detectable aggregates and that labeled Fc retained pH-dependent binding to FcRn. Nanogold : Fc ratios determined spectrophotometrically and estimated by SDS-PAGE were typically 0.81.1, suggesting most Fc molecules were singly labeled.

FcRn-coupled liposomes were incubated either with unlabeled Fc fragments or with Nanogold-labeled Fc under similar conditions. After 30 minutes at room temperature, 2 µL of each sample were mixed with 2 µL of 10 nm colloidal gold (as fiducial markers for alignment), then loaded onto glow-discharged Lacey-carbon grids and plunge-frozen in liquid ethane at liquid nitrogen temperature. For two-dimensional (2D) imaging, cryoEM was performed on a FEI Tecnai 12 microscope at 120 kV at 2 mm defocus, with a dose of ~20e-/Å2 per image, and images were recorded on a 2X X 2K CCD at a magnification of 42,000 X. For tomographic reconstructions, tilt series (-60° to +60° 1.5° step size) were collected using a FEI Tecnai F30 microscope operating at 300 kV and controlled by a software package developed at University of California, San Francisco. The defocus was set to 4.58.0 µm at 0°. Images were recorded with a 2K X 2K CCD at a magnification of 34,000 X with a total dose of 80100 e-/Å2 for each tilt series. The IMOD software package was used for data processing and 3D reconstructions. A total of 23 independent tilt series were collected and reconstructed.

        

[Nanogold labeling reagents and structure of Nanogold-Fc-FcRn liposomes (81k)]

Upper: Nanogold labeling reagents: Monomaleimido- Nanogold and Mono-Sulfo-NHS-Nanogold, showing mode of reaction and biomolecule labeling. Lower: Binding of Nanogold-labeled Fc to FcRn-liposomes. Residue 19 of the FcRn light chain (beta-2-microglobulin), which was mutated to cysteine in the K19C FcRn mutant, is highlighted as a red sphere linked to the liposome surface.

FcRn is normally a type I membrane protein composed of a heavy chain with a single transmembrane spanning region and a noncovalently attached soluble light chain (beta-2-microglobulin). In the absence of Fc, most of the liposomes were shown to be isolated, spherical structures with diameters of 50 - 200 nm. However, upon addition of purified rat Fc, the liposomes showed increased aggregation, with visible density between adjacent lipid bilayers and flattened interfacial contacts between adjacent liposomes. Their inner vesicles (which would not have access to FcRn or Fc) remained spherical. FcRn can bind to both chains of a homodimeric Fc molecule to form a 2 : 1 FcRn/Fc complex, and these results suggested that the addition of Fc caused bridging between adjacent FcRn-coupled vesicles through the binding of FcRn molecules on separate vesicles to the same Fc dimer. ECT was then used to generate 3D reconstructions of adhering liposomes: in the resulting tomograms, most of the interfaces between two adjacent lipid membranes showed continuous densities between bilayers, with a typical distance between adjacent membranes in an interface of ~150 Å, consistent with the width of a 2 : 1 FcRn-Fc complex; however, the limited resolution of the tomograms did not permit conclusive identification of individual complexes. To gain more information, the ECT imaging was repeated using Nanogold-labeled Fc. Clusters appeared as dark densities, each ~2 nm in diameter, corresponding to the diameter of a single Nanogold cluster plus ligands. These dark densities were often in rows of 2 to 20 at the center of a liposome-liposome interface. Cluster densities were separated by at least ~8 nm, consistent with the closest distances between adjacent FcRn-Fc complexes. These results suggest that cluster densities represented singly-labeled Fc molecules bridging between FcRn proteins coupled to adjacent liposomes. When Nanogold-labeled Fc was visualized in ice in the absence of FcRn-coupled liposomes, the gold clusters were randomly distributed.

These results demonstrate that the 1.4 nm Nanogold cluster is visible in tomograms of typically thick samples (250 nm) recorded with defocuses appropriate for large macromolecules, and is thus an effective marker for this method which overcomes the thickness limitations previously associated with small gold labels for electron tomography. Furthermore, Nanogold was covalently attached to the reduced sulfhydryl of the Fc hinge region, a site distant from the FcRn binding site, and the resulting Nanogold-labeled Fc functioned normally in FcRn binding and uptake into cells; this approach is therefore appropriate for preparing probes for these experiments.

Reference:

  • He, W.; Jensen, G. J., and Björkman, P. J.: Nanogold as a Specific Marker for Electron Cryotomography. Microsc. Microanal., 15, 183-188 (2009).

Reference for Fc preparation:

  • Martin, W. L. & Björkman, P. J.: Characterization of the 2:1 complex between the class I MHC-related Fc receptor and its Fc ligand in solution. Biochem., 38, 1263912647 (1999).

More information:

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Best Approaches to Membrane Blotting and Lateral Flow Assays

If you have been using Nanogold® for detection on nitrocellulose membrane blots (immunodot blots or Westerns) and experienced less than ideal detection, we have developed an optimized detection procedure that maintains the already very high sensitivity, but combines it with a greatly reduced background and enhanced signal clarity. For the best results, we recommend using a procedure that incorporates the following features:

  • Use gold enhancement rather than silver enhancement to develop the signal after application of the Nanogold conjugate. In most cases, this will provide substantially lower background or non-specific signal, while maintaining a similar level of sensitivity to silver enhancement.

  • Incorporate 0.1% Tween-20 (detergent) in the buffers used for blocking, antibody incubation, and washing. This will dramatically reduce background binding.

  • Include 1% nonfat dried milk (you can use the material sold in supermarkets and food stores) as an additive in the incubation buffer (the buffer in which the Nanogold is dissolved and applied to the blot) and 5% nonfat dried milk in the blocking buffer used to block the membrane before application of antibodies.

Suggested procedure:

REAGENTS AND EQUIPMENT:

  • Phosphate buffered saline (PBS): 20 mM sodium phosphate buffer pH 7.4 and 150 mM NaCl.
  • Specific antigen (target protein or other biomolecule).
  • Nitrocellulose (NC) membrane 0.2 µm pore size.
  • Blotting Paper to wick membrane dry.
  • Orbital Shaker
  • Washing buffer (TBS-Tween 20): 20 mM Tris pH 7.6, with 150 mM NaCl and 0.1 % Tween-20.
  • Nonfat dried milk (Carnation)
  • GoldEnhance EM (Nanoprobes Product No. 2113).
  • Specific Nanogold antibody conjugate.

PROCEDURE:

Antigen Application:

  1. Prepare antigen solutions with a series of dilutions (0.01mg/mL, 0.001mg/mL, 0.0005mg/mL, 0.0001 mg/mL, 0.00005 mg/mL, 0.00001 mg/mL and 0.000005 mg/mL) using PBS, pH7.4.
  2. Pipette 1 µL of above solutions to a dry nitrocellulose membrane; prepare 2 duplicates as a negative control.

    • Negative control 1: No antigen, No antibody.
    • Negative control 2: No antigen with NG-conjugate incubation.

  3. Air-dry for 30 minutes

Blocking:

  1. Immerse membranes in 8 mL of TBS-Tween 20 for 5 minutes.
  2. Block membranes in 8 mL of TBS-Tween 20 containing 5 % nonfat dried milk for 30 minutes at room temperature.

Binding of Nanogold antibody conjugate:

  1. Dilute Nanogold antibody conjugate in TBS-Tween20 containing 1% nonfat dried milk to 4 µg/mL (1:20 Dilution: 300 µL conjugate + 5.30 ml TBS-gelatin containing 1% nonfat dried milk).
  2. Incubate the membranes in 8 mL of diluted conjugate solution for 30 minutes at room temperature.
  3. Incubate the control membrane in 8 mL of TBS-Tween20 containing 1% nonfat dried milk for 30 minutes at room temperature.

Autometallographic Detection:

  1. Wash membranes three times for 3 min each in 8 mL of TBS-Tween 20. Wash membranes thoroughly in 8 mL of deionized water (4 x 3 minutes). Make sure strips are washed separately according to what they are incubated in (strips incubated in one lot of a conjugate are washed in a separate dish from strips that are incubated in TBS-Tween 20 with 1% nonfat dried milk without conjugate, strips incubated in different lots are washed separately).
  2. Perform Gold Enhancement according to instructions (mix solutions A and B, wait 5 minutes, then add C and D).
  3. Record the number of observed spots and time when the spots appear. Record the time when background appears on the control membrane.
  4. After 15 minutes, the enhancement solution is removed. Rinse membranes with water (3 x 3 minutes) and air-dry for storage.

Tween-20 and nonfat dried milk should also improve the performance of Nanogold conjugates used with silver enhancement.

Examples of detection with Nanogold on blots are shown below, in comparison with detection using conventional colloidal gold.

[Nanogold and colloidal gold blots illustrating detection sensitivity (63k)]

Blot test with Nanogold and colloidal gold conjugates. (a) dot blot image of Nanogold-Fab' Goat anti-Rabbit IgG, developed with GoldEnhance EM gold enhancement reagent, using the Schleicher & Schuell (recently acquired by Whatman) MINIFOLD-1 Dot Blot System which applies the target precisely over a circle with a diameter of 5 mm. The 5 mm circle is big enough for a quantitative analysis by a densitometer. With the target spreading over 5mm circle, we can detect as low as 0.01 ng antigen. While we use a pipette to apply 1 L of target over a circle of 1-2 mm, we can detect as little as 0.005 ng by the same NG conjugates. (b) Nanogold anti-mouse Fab' blotted against mouse IgG, developed with LI Silver (Nanoprobes), showing sensitivity enhancement with smaller spot size. Target was applied using a 1 µL microcapillary tube. This immunodot blot shows 0.1 pg sensitivity (arrow). (c) Colloidal gold blot using a 15 nm colloidal gold conjugate to detect serial dilutions of an IgG target, without silver or gold enhancement. The range of color densities should give a good basis for comparing your results - i.e. if your spot is darker than our 50 ng spot your conjugate is working very well, but if it is much fainter, it is questionable.

Highly colored particles, including colloidal gold, are used to generate the colored signals in immunochromatographic lateral flow devices - the "rapid tests" that you might use to detect pregnancy, or diagnose an infection quickly in a doctor's office.

In general, the colloidal gold particles used for immunochromatographic devices are much larger than our products. Our Nanogold® particles are 1.4 nm in diameter; the colloidal gold used for immunochromatographic assays is usually 40 nm in diameter or larger. These larger particles are much more highly colored than Nanogold: consequently, relatively few are needed to form a signal that can be seen by eye once they have been captured or bound to a target on an immunochromatographic device.

Nanogold, because of its smaller size, requires autometallographic enhancement for visualization if it is used in these applications. Nanoprobes offers both silver enhancers and gold enhancement which may be used to develop Nanogold for optical observation. Once it has been developed with these reagents, Nanogold can provide significantly enhanced sensitivity: in dot blots, silver-enhanced Nanogold has demonstrated improvements of an order of magnitude or more over colloidal gold.

The simplest approach is to "develop" the device by applying the mixed reagents to it after it has been run and the gold-labeled antibody has bound to its target. However, the fabrication of an assay device in which a silver salt for silver enhancement is incorporated into the device itself, and activated when the sample is applied, has been described in the literature. The construction of such a device, and a comparison with a conventional colloidal gold lateral flow device, is shown below.

Construction of lateral flow immunochromatographic devices using colloidal gold and silver-enhanced gold [(53k)]

Construction of a lateral flow device using colloidal gold (left), and colloidal gold with silver enhancement (right).

The preparation of gold conjugates and the construction of immunochromatographic assays is a complex field in which much of the critical information is difficult to find or not publicly available. One reference which serves as a good introduction is found in IVD Technology magazine:

Because this application has not been extensively investigated with Nanogold, we do not offer a specific procedure for making Nanogold conjugates for immunochromatographic applications. We recommend antibody labeling with Monomaleimido Nanogold according to our usual protocols; the conjugate product may then be incorporated into immunochromatographic devices in the same manner as larger colloidal gold conjugates.

Reference for silver-enhanced lateral flow assay device:

Nanoprobes, Incorporated, currently does not manufacture colloidal gold. However, we have continued to make our technical support page for colloidal gold and its conjugates available as general information to the community. We are currently working to develop larger gold labeling reagents and conjugates with the same covalent, site-specific reactivity as our smaller Nanogold compounds, and have published a number of preliminary results from this work.

More information:

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AuroVistTM: Tips for Improved X-Ray Contrast

You may already know about AuroVistTM, the first gold nanoparticle X-ray contrast agent for micro CT and CT imaging in research applications. AuroVistTM provides contrast enhancement up to ten times that of iodine-based reagents. You can obtain high-resolution, high-contrast images of blood vessels, organs, other anatomical structures and tumors in animals. AuroVistTM is highly soluble, biocompatible, and stable to the environment found in the vascular system and in tissues. Unlike many iodine reagents, it also has very low viscosity and osmolality. This means less traumatic injections, and the ability to inject into smaller blood vessels with much lower risk of damage.

AuroVistTM is a stabilized 1.9 nm gold particle. It provides better contrast than iodine for both micro-CT and clinical CT applications.* At appropriate beam energies (typically just above the gold L and K edges), gold achieves a contrast up to three times greater than iodine per unit mass, yielding initial blood contrast greater than 500 Hounsfeld Units (HU). When combined with achievable gold concentrations up to four times those of iodine, this provides a total contrast gain of up to ten times or more. AuroVistTM gives you these enhanced performance features:

  • Longer blood residence time than iodine agents, due to its larger size (1.9 nm gold core, ~50,000 Da).

  • High contrast (>500 HU initial in blood, kidneys >1,500 HU).

  • Clears through kidneys. Kidney fine structure may be imaged from 1-2 minutes up to an hour or more after injection; concentration in the kidneys can provide contrast values as high as 1,500 HU or more.

  • Permeates angiogenic endothelium, enabling imaging of tumors.

  • Concentration >4 times that of standard iodine agents (up to 1.5 g Au/cc).

  • Can be imaged using standard microCT.
  • Low toxicity (LD50 >1.4 g Au/kg).

  • Up to 10 times the contrast of standard iodine agents (Gold absorbs ~3 times more than an equivalent weight of iodine at 20 and 100 keV and can be ~4 times more concentrated, giving more than 10-fold combined contrast enhancement.

  • Low osmolality, even at high concentrations

  • Low viscosity, similar to water; easy to inject, even into small vessels.

  • Yields enhanced radiotherapy dose.

AuroVist mouse, kidney and inferior vena cava images [(104k)]

(Upper left): Live mouse, 5 minutes after injection. (Upper right) Imaging of kidney in live mouse 1 hour after injection, showing kidney contrast and fine structure (bar = 1 mm). (Lower left): Live mouse, 2 minutes after injection showing vascular fine structure (bar = 5 mm); (Lower right) MicroCT of mouse inferior vena cava (bar = 1 mm).

* Research use only. Not approved for clinical or human use.

AuroVistTM is a new product, and therefore it has not been optimized in all possible applications. However, the following guidelines may be helpful in obtaining the best results with this reagent.

Best instrument and beam settings

The absorption increases by a significant factor (jump ratio) above the gold L and K edges. The X-ray properties of gold, showing the jump ratios for these regions, are shown in the table below. It is therefore advantageous to image using these absorptions. The settings below are appropriate for the different instruments.

This must be compared this with the absorption spectrum of soft tissue. X-ray absorption spectra for elements and tissues are available from NIST:

X-ray absorption spectra

  Energy (keV) µ/p(cm2/g) jump ratio
  11.8 75.8  
L3 11.9 187.0 2.5
  13.6 128.3  
L2 13.7 176.4 1.4
  14.3 158.8  
L1 14.4 183.0 1.2
  80.6 2.1  
K 80.7 8.9 4.2
  • Mammography: These instruments are suitable for small animal imaging. Use of lower kVp, at or below the L-edge absorptions of gold at ~13 keV, or ~40 kVp (e.g., 22 kVp) is recommended to take advantage of the L edge gold absorptions. Exposures are typically 1 second or less for a mouse, so live imaging is possible. Resolution can be < 0.1 mm.

  • Clinical CT: 80 kVp gives the greatest attenuation, but higher voltages, particularly with filtering can make use of the Au K edge; beam energy can be tuned to just above gold's 80.7-keV K-edge (typically 120 kVp or higher). Imaging time is typically a few seconds, with resolution ~0.3 mm.

  • MicroCT: Here the resolution is increased (to even 2 microns), but the tube power is typically ~100 times less than a clinical unit. Fine area 2D detectors mean that many tiny pixels must each receive enough counts. This then requires a much longer imaging time (e.g., 0.5 - 2 hours) than clinical CT. Many units also slow the tube rotation such that only 1 revolution is done in the selected imaging time (e.g., 1 hour). Animal movement must be minimized during this time. One solution is to sacrifice the animal, but live imaging has been accomplished if the region can be gated or immobilized during the imaging time. Beam energy should be just above golds 12-14-keV L-edge. To enhance detection, or give the best absorptive contrast, the ideal settings are those that overlap the L-edge absorptions of gold at ~13 keV; this requires a kVp of about 3 times this or ~40 kVp - so the first choice for kVp setting would be 40 kVp, or as close as the instrument allows.

How to ensure minimal toxicity

Certain strains of mice appear to be more tolerant of this gold. For Balb/C, the LD50 is ~ 3.2 g Au/kg. Nude mice and C3H mice also seem to respond about the same. Some outbred mice, however, appear to have a lower LD50 of about > 1.4 g Au/kg. If you are using outbred mice, or a different strain to those mentioned above, it is recommended that you use this lower value. The following suggestions may be helpful:

  • Start with a moderate dose, such as 120 or 160 mg/mL (for 0.2 mL of solution). The value of 270 mg/mL reported in our paper in the British Journal of Radiology was the highest value tested; at high concentrations acute toxicities increase rapidly, and a modest reduction in dose can significantly reduce toxicity without compromising your results.

    The average mouse has a mass of about 20 g, and a circulatory system volume of about 1 mL. The amount of AuroVist necessary to achieve some of the suggested g Au/kg values is as shown below:

    Mouse body mass Gold dose, g Au/kg Amount of AuroVist required, mg Injection volume at 200 mg Au/mL
    20 g 0.28 5.6 28 µL (0.028 mL)
    20 g 0.7 14.0 70 µL (0.07 mL)
    20 g 1.4 28.0 140 µL (0.14 mL)
    20 g 2.0 40.0 200 µL (0.2 mL)
    20 g 3.2 64.0 320 µL (0.32 mL)*

    * Use more concentrated solution: keep injection volume to 0.2 mL or less.

  • Centrifuge and then filter the reconstituted reagent through a sterile filter immediately before injection to ensure that no aggregates or larger particles are present. Larger particles and aggregates can impact biocompatibility, and may also have reduced stability and hence a higher tendency to deposit in tissues and organs, with negative consequences.

  • Use one of the pure-bred mouse strains mentioned above, which are known to have higher tolerance. If you are planning to conduct multiple experiments using a different strain, it may be worthwhile to test your strain first.
We encourage you to tell us how well it works in your application, or if you encounter problems; that way, you can help contribute to the knowledge base on this reagent and its applications.

We have received several inquiries about whether a targeted version of this reagent is available. The preparation of a targeted gold nanoparticle reagent on a sufficient scale for X-ray contrast imaging is challenging both because of the greater complexity of the synthesis, and also because of the amount of reagent required for visualization. This depends on the size and target density of the feature to be imaged, and also on how effectively the targeting mechanism can deliver a visible dose to the target, and this may be less than preliminary studies such as blots or cell studies suggest. Other studies described in the literature indicate that the targeting methods investigated so far direct at most 15 - 25% of the reagent to the target site. However, we are working to develop this technology, and hope to incorporate it into future AuroVistTM products.

References:

  • Hainfeld, J. F.; Slatkin, D. N.; Focella, T. M, and Smilowitz, H. M.: Gold nanoparticles: a new X-ray contrast agent. Br. J. Radiol., 79, 248-253 (2006).

  • Hainfeld, J. F.; Slatkin, D. N.; Focella, T. M., and Smilowitz, H. M.: In Vivo Vascular Casting. Microsc. Microanal., 11, (Suppl. 2: Proceedings); 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, p. 1216CD (2005).

  • Hainfeld, J. F., Slatkin, D. N., and Smilowitz, H. M.: The use of gold nanoparticles to enhance radiotherapy in mice. Phys. Med. Biol., 49, N309-N315 (2004).

More information:

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Nanogold® and the Role of Syntaxin-18

Nanogold® labeling with gold enhancement provides one of the most precise methods for the localization of targets. Gold enhancement is an alternative to silver enhancement, developed by Nanoprobes. Gold enhancement works similarly to silver enhancement, except that it is gold - instead of silver - that is deposited onto colloidal gold or gold cluster labels. This catalytic enlargement and enhancement process produces enlarged particles for electron microscopic observation, and dark staining for light microscopy, blotting and optical applications. It has important advantages over silver enhancement for several applications:

  • Cleaner signals with lower background for light microscopy and blotting.
  • Osmium etch resistance for EM: may safely be used before any strength osmium tetroxide.
  • Compatible with physiological buffers: does not precipitate with halides.
  • Compatible with metal substrates for cell culture or biomaterials.
  • Less pH sensitive than silver enhancement: can be used in a wider pH range.
  • Better for SEM Visualization: much stronger backscatter signal than silver.
  • Near neutral pH for best ultrastructural preservation. Low viscosity for ease of use.

[GoldEnhance: mechanism and EM labeling (84k)]

(left) Size comparison of Nanogold-Fab' with conventional 5 nm colloidal gold-IgG probe, showing overall probe size and distance of gold from target. (right) Pre-embedding immunolabeling using Nanogold-Fab and GoldEnhance EM, showing uniform enlarged particles (see: Marra, P.; Salvatore, L.; Mironov, A Jr.; Di Campli, A.; Di Tullio, G.; Trucco, A.; Beznoussenko, G.; Mironov, A., and De Matteis, M. A.; Mol. Biol Cell., 18, 1595-1608 (2007)).

The endoplasmic reticulum (ER) in mammalian cells is a reticular tubular network that extends from the nucleus to the cell periphery along the microtubule track. The presence of subdomains in the ER enables this organelle to perform a variety of functions, yet the mechanisms underlying subdomain organization are poorly understood. In a recent study reported in the Journal of Cell Science, Iinuma and group used electron microscopy with gold-enhanced Nanogold labeling to help demonstrate that syntaxin 18, a soluble NSF attachment protein (SNAP) receptor localized in the ER, is important for the organization of two ER subdomains, smooth/rough ER membranes and ER exit sites. To examine the function of syntaxin 18, the authors knocked down its expression in HeLa cells using two short interfering RNAs (siRNAs), then examined the resulting ER structures using electron microscopy to examine the effects at the ultrastructural level.

Conventional and immunoelectron microscopy showed that Immunoelectron microscopy confirmed Golgi disassembly and the formation of ER membrane aggregates in syntaxin-18-depleted cells. The pre-embedding gold enhancement immunogold method was used for immunoelectron microscopy. Cells were cultured on plastic coverslips and were fixed in 4% paraformaldehyde in phosphate buffer (PB) for 2 hours at room temperature. After permeabilization in PB containing 0.25% saponin for 30 minutes, followed by blocking for 30 minutes in PB containing 0.1% saponin, 10% bovine serum albumin, 10% normal goat serum and 0.1% cold water fish skin gelatin, the cells were incubated overnight with rabbit or mouse primary antibodies in the blocking solution. The specimens were incubated with Nanogold-conjugated goat anti-rabbit IgG or mouse-IgG in blocking solution for 2 hours; the signal was then intensified using GoldEnhance EM for 3 minutes at room temperature. The specimens were post-fixed in 1% OsO4 containing 1.5% potassium ferrocyanide and were processed for electron microscopy by washing with distilled water, dehydrated in a series of graded ethanol solutions and embedded in epoxy resin for electron microscope observation.

Knockdown of syntaxin 18 caused a global change in ER membrane architecture, dispersion of the Golgi apparatus, and segregation of the smooth and rough ER. Furthermore, the organization of ER exit sites was markedly changed with dispersion of the ER-Golgi intermediate compartment and the Golgi complex. These morphological changes in the ER were substantially recovered by treatment of syntaxin-18-depleted cells with brefeldin A, a reagent that stimulates retrograde membrane flow to the ER. These results suggest that syntaxin 18 has an important role in ER subdomain organization by mediating the fusion of retrograde membrane carriers with the ER membrane.

Reference:

  • Iinuma, T.; Aoki, T.; Arasaki, K.; Hirose, H.; Yamamoto, A.; Samata, R.; Hauri, H. P.; Arimitsu, N.; Tagaya, M., and Tani, K.: Role of syntaxin 18 in the organization of endoplasmic reticulum subdomains. J. Cell. Sci., 122, 1680-1690 (2009).

More information:

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Reach the Right Department: Contact Information

If you are calling or e-mailing us, you should note that not all of our contacts have access to all your (or our) information. Therefore, it is worth taking a moment to make sure that your question will go to the person who can find the information you are looking for.

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. You should not contact 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, or fill out our custom synthesis request form. 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

If you are looking for a specific person, our web site includes a listing of key personnel with individual extensions and e-mail addresses.

In June, we welcome back John Dubendorff in the position of Research Scientist. He will be working to develop novel gold labeling methods and technology, and alternative approaches to probes and labeling.

More information:

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Other Recent Publications

As we have discussed previously, silver-enhanced Nanogold® labeling may be combined with peroxidase labeling for a double labeling immunoelectron microscopic method in which the two targets are distinguished by stains with entirely different characteristics. In a recent paper in Cerebral Cortex, Huo and group provide a useful demonstration of the method. Their study examined morphological connections of GABAergic (gamma-aminobutyric acidergic) neurons and serotonergic projection terminals from the dorsal raphe nucleus (DR), and the relationship between GABAergic terminals and ventrolateral orbital cortex (VLO) neurons projecting to the PAG, using anterograde and retrograde tracing combined with immunofluorescence, immunohistochemistry, and electron microscopy methods. Double immunoelectron microscopy was used to investigate the localization of GABA and 5-HT1A (5-hydroxytryptamine 1A) receptor in 50 µm rat forebrain sections containing VLO.

For GABA/5-HT1AR dual label staining, the sections were incubated for 24 hours at room temperature with a mixture of rabbit antiserum against GABA polyclonal antibody (A2052, 1:1000 dilution) and guinea pig antiserum against 5-HT1AR polyclonal antibody (AB5406, 1:1000 dilution) in TBS-NGS, then incubated with Nanogold anti-guinea pig IgG (1:100 dilution) and biotinylated goat anti-rabbit IgG (1:100 dilution) overnight at room temperature. After incubating, the sections were washed in 0.05 M TBS 3 times and subsequently processed as follows:

  1. postfixation with 1% glutaraldehyde in 0.1 M phosphate buffer (PB) for 10 minutes;
  2. after washing in distilled water, silver enhancement with HQ Silver in the dark;
  3. incubation with ABC Elite Kit (Vector) diluted at 1:100 in 0.05 M Tris-buffered saline for 2 hours at room temperature;
  4. visualization of 5-HT-like immunoreactivity by incubating with 0.02% (w/v) DAB and 0.0003% H2O2 for 20-30 minutes;
  5. osmification with 1% osmium tetroxide (OsO4) in 0.1 M PB at room temperature for 45 minutes;
  6. counterstaining with 1% uranyl acetate in 70% ethanol in the dark for 1 h; and finally
  7. flat-embedding in Durcupan after dehydration in a graded series of ethanol, and mounting on silicon-coated slide glass.

Results indicate that the majority (93%) of GABAergic neurons in the VLO also express the 5-HT1A (5-hydroxytryptamine 1A) receptor, and serotonergic terminals originating from the DR nucleus made symmetrical synapses with GABAergic neuronal cell bodies and dendrites within the VLO. GABAergic terminals also made symmetrical synapses with neurons expressing GABAA receptors and projecting to the PAG. These results suggest that a local neuronal circuit of 5-HTergic terminals, GABAergic interneurons, and projection neurons, exists in the VLO, and provides morphological evidence for the hypothesis that GABAergic modulation is involved in 5-HT1A receptor activation-evoked antinociception.

Reference:

  • Huo, F. Q.; Chen, T.; Lv, B.-C.; Wang, J.; Zhang, T.; Qu, C. L.; Li, Y. Q., and Tang, J. S.: Synaptic connections between GABAergic elements and serotonergic terminals or projecting neurons in the ventrolateral orbital cortex. Cereb. Cortex, 19, 1263-1272 (2009).

Probes and methods for pre-clinical molecular imaging for oncology are reviewed by Mather in a recent paper in Bioconjugate Chemistry. The definition of molecular imaging provided by the Society of Nuclear Medicine is "the visualization, characterization and measurement of biological processes at the molecular and cellular levels in humans and other living systems." This review gives an overview of the technologies available for and the potential benefits from molecular imaging at the preclinical stage; it includes both the biological considerations of targeting methods, and the design of probes for imaging using different visualization techniques. The article focuses on the use of imaging probes based on bioconjugates for applications in the field of oncology; however, molecular imaging can be equally useful in many fields, including cardiovascular medicine, neurosciences, infection, and others.

Reference:

  • Mather, S.: Molecular Imaging with Bioconjugates in Mouse Models of Cancer. Bioconj. Chem., 20, 631-643 (2009).

We are always interested to learn about alternative imaging methods, and Bertin and colleagues recently reported the use of a dendritic manganese-based contrast agent for MRI imaging in a recent paper in Bioconjugate Chemistry. MRI contrast enhancement depends upon the presence of unpaired electrons, and manganese is an ideal candidate since the Mn2+ ion has five unpaired electrons. The new reagents were based on diethylenetriamine pentaacetic acid, linked to an aromatic polyether derivative for use as a brain imaging reagent. The new reagent displayed high hydrosolubility and no in vitro neuronal toxicity at concentrations as high as 1 mM. T1 relaxivity of 4.2 mM-1.s-1 for the manganese compound and T2 relaxivity of 17.4 mM-1.s-1 for its gadolinium analog at 4.7 T were measured: these are higher than that of the commercial MRI contrast agents GdDTPA and MnDPDP, respectively.

Reference:

  • Bertin, A.; Steibel, J.; Michou-Gallani, A. I.; Gallani, J. L., and Felder-Flesch, D.: Development of a dendritic manganese-enhanced magnetic resonance imaging (MEMRI) contrast agent: synthesis, toxicity (in vitro) and relaxivity (in vitro, in vivo) studies. Bioconj. Chem., 20, 760-767 (2009).

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