N A N O P R O B E S     E - N E W S

Vol. 8, No. 7          July 27, 2007

Enzyme Metallography is a biological labeling and staining method in which a targeted enzymatic probe is used to selectively deposit metal at sites of interest. It has proven to very clean and sensitive both for in situ hybridization, where it readily visualizes endogenous copies of single genes, and immunohistochemistry (IHC) detection. In the electron microscope, it produces dense, highly specific staining with very high penetration. It has also been applied successfully as an electrical detection method for biochips: this application, in which multiple independent electrical contacts are fabricated upon target binding, offers the potential for highly multiplexed target detection in a robust, miniaturized and highly portable format.

The sharply resolved black signal, used for in situ hybridization, is readily distinguished from other stains, and has been combined with fast red K immunohistochemistry to provide a concomitant brightfield gene and protein assay (EnzMet GenePro). Enzyme metallography has many advantages as a detection method. Because it is used in the conventional brightfield electron microscope, it does not require either expensive fluorescent optics, or dark adaptation on the part of the user. The signal is permanent, and does not have the photobleaching problems associated with fluorescent stains. Furthermore, because the deposited metal is electron-dense, it provides high contrast for electron microscopy, making enzyme metallography both a highly effective electron microscopic method, and a potential correlative light and electron microscopic method.

Nanoprobes recently signed a deal with Ventana Medical Systems, Incorporated, for the commercial development and use of this reagent in automated slide staining instruments and applications. 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. Nanoprobes will shortly introduce a commercial EnzMetTM formulation optimized for research applications and non-automated staining. Look for an announcement on our web site later this year.

Reference:

• Powell, R. D.; Pettay, J. D.; Powell, W. C.; Roche, P. C.; Grogan, T. M.; Hainfeld, J. F., and Tubbs, R. R.: Metallographic in situ hybridization. Hum. Pathol., 38, 1145-1159 (2007).
  • Abstract (courtesy of Science Direct).

More information:

• Enzyme Metallography for light and electron microscopy of Microsporida (Microscopy & Microanalysis 2006 abstract)
• Enzyme Metallography for correlative labeling (Microscopy & Microanalysis 2004 abstract)
• Enzyme Metallography - a New staining method (Microscopy & Microanalysis 2002 abstract)
• Product application special feature: Nanogold in situ hybridization
• Other product and research applications

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Nanogold® Labeling: Filtration, Purification, and Beating Aggregation

Filtration

If you need to use an antibacterial filter (for example, to prepare a sterile solution), we recommend cellulose acetate rather than nylon or other filters. for final purification and packaging of Nanogold conjugates, we routinely use Nalgene 0.2 micron cellulose acetate syringe filters. We have occasionally observed undesirable interactions between Nanogold conjugates and other filter materials: in the case of our customer, it appears that the Nanogold bound strongly to the nylon filter material and was partially degraded, leading to cleavage of the conjugate protein and the observed labeling reduction.

If your Nanogold conjugate becomes stuck on the filter, you may be able to redissolve it by using DMSO (dimethyl sulfoxide). DMSO is a highly effective solvent for Nanogold: use in a mixture with water at the maximum proportion of DMSO that (a) your conjugate biomolecule, and (b) your filter material can tolerate. Many antibodies and proteins will tolerate 20% DMSO, and some peptides and small molecules much higher proportions, while many filter materials can tolerate up to 70%.

Liquid Chromatography - Gel filtration: how to recognize and beat aggregation

One explanation proposed for our customer's result was that they were actually separating an aggregated Nanogold species from unlabeled material. This is unlikely, because the calculated labeling indicated close to a 1 : 1 ratio of Nanogold to antibody, and the material eluted from the column as a reasonably well-defined peak. But this raises the question: how do you recognize and beat aggregation?

Below, we show what a successful separation looks like when a Nanogold-labeled protein (in this case antibody Fab' fragments) is separated from excess Nanogold over a gel filtration column.

How will you know when you have aggregation?

• When Nanogold aggregates, there may be a color change: the gold can form larger particles that are red or red-brown, or may precipitate to give a gray or blue-black precipitate.
• Aggregates often stick at the top of the column. Look for a brown or black discoloration at the top of the column that does not move with the eluent.
• Aggregated materials are generally larger than the protein-Nanogold conjugate. They will tend to elute near or at the column void volume if they make it through the column.
• If aggregation occurs, the resulting particles tend to form a range of sizes. If part of this range overlaps with the size fractionation range of the column, they will tend to give a broad and ill-defined peak.

How can you avoid it?

• When you separate your Nanogold conjugate after the conjugation reaction, use a column material with a separation or fractionation range that extends significantly above the expected size of the conjugate. Should larger species be formed, they will then be separated from the conjugate. Below is a table listing some of the chromatography media that are available and their separation ranges; check these and published separation ranges for other materials to find an appropriate material.

• Sometimes precipitation can result from solubilization problems. In this case, use a proportion of DMSO (dimethyl sulfoxide) in the reaction buffer and the column elution buffer. Most proteins can tolerate up to 20% DMSO. However, use caution when adding DMSO as mixing with water is exothermic, and the heat generated can accelerate hydrolysis of the reactive group. Cool buffers in the refrigerator before mixing.

• You may need to vary the reaction conditions in order to achieve the cleanest reaction. In this case, it is sometimes helpful to order the Nanogold labeling reagent in five aliquots rather than 30 nmol in one vial, so you can run two or three different reactions under different conditions.

We have found that the following media work well for gel filtration of different Nanogold-labeled conjugates:

More information:

• Guide to Gold Cluster Labeling
• Guide: Separating and Isolating Gold Cluster Conjugates
• Guide: Spectra and Labeling Calculation
• Guide: Advanced Labeling Calculation (colored biomolecules)
• Plan your labeling: separation methods and strategies (previous article)
• Technical Help Nanogold labeling reagents
• Catalog information - Nanogold labeling reagents
• References - Nanogold Labeling

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NTA-Ni(II)-Nanogold® Reveals Lipoprotein-Secretin Interactions

Neisseria meningitidis is a causative agent of meningitis and septicemia. It is a bacterium which expresses type IV pili, which mediate functions including autoagglutination, twitching motility, biofilm formation, adherence, and DNA uptake during transformation. The secretin PilQ supports type IV pilus extrusion and retraction, but also requires auxiliary proteins for assembly and localization in the outer membrane. In the current issue of the Journal of Bacteriology, Collins and group describe the use of NTA-Ni(II)-Nanogold labeling of a His-tagged PILP fusion protein to study the physical properties of the lipoprotein PilP and its interaction with PilQ. Results from solid phase overlay assays, mutagenesis and blotting studies showed that PilP was an inner membrane protein, required for pilus expression and transformation, since pilP mutants were nonpiliated and noncompetent. In vitro experiments using recombinant fragments of PilP and PilQ showed that the N-terminal region of PilP interacted with the middle part of the PilQ polypeptide, and NTA-Ni(II)-Nanogold labeling and cryoelectron microscopy were then used to elucidate the structure of the complex.

A meningococcal in-frame pilP-His fusion was designed to encode a six-histidine tag in the predicted loop region at amino acid 74. The PilQ complex was purified from meningococcus outer membranes by detergent solubilization and size exclusion. For native PilP purification, meningococcus M1080-PilP74-6xHis cells were grown on blood agar plates in 5% CO₂ at 34°C overnight, harvested in cold phosphate-buffered saline (PBS) and collected by centrifugation at 4,000 x g for 15 minutes. The cell pellet was resuspended in cold Tris-buffered saline (pH 7.5) containing 1 x EDTA-free protease inhibitor cocktail and passed twice through a French press at 25,000 lb/in². Cell debris was removed by centrifugation twice at 4,000 x g for 10 minutes. The supernatant was collected and membrane fraction pelleted by centrifugation at 35,000 rpm for 35 minutes. The pellet was dissolved in lysis buffer (10 mM imidazole, 300 mM NaCl, 50 mM NaH₂PO₄, 0.5% Triton X-100, 10 mM beta-mercaptoethanol, 3 mM MgCl₂, pH 8.0) with 1 x EDTA-free protease inhibitor cocktail and incubated with rotation at 4°C overnight, then the clear fraction was centrifuged at 4,000 x g for 15 minutes. The supernatant was purified over a Ni-NTA agarose column using elution buffer (200 mM imidazole, 300 mM NaCl, 50 mM NaH₂PO₄, pH 8.0) then immediately dialyzed against 50 mM sodium phosphate buffer (pH 7.0) containing 300 mM NaCl and 10% glycerol.

Natively purified recombinant PilPDelta₁₁₉ (100 µg/mL) was incubated with PilQ complex purified from outer membranes, with or without NTA-Ni(II)-Nanogold with agitation for 24 hours at 4°C, then centrifuged at 13,000 rpm in a benchtop centrifuge for 5 minutes and prepared for cryonegative staining. Grids were placed in a cryostage, and data recorded at 100 K in conjunction with a 4K charge-coupled-device (CCD) camera. Individual gold-labeled particles were interactively selected in 64-pixel boxes (Å/pixel 3.1) using BOXER. After contrast transfer function correction, the data were contrast normalized, centered, and low-pass filtered to 25-Å resolution. A 190-Å circular mask was applied, six rounds of iterative refinement performed in C4 symmetry using the previously calculated 3D structure of PilQ filtered to a 25-Å resolution as a start model.

The three-dimensional reconstruction of the PilQ-PilP interacting complex obtained at low resolution by transmission electron microscopy showed that PilP localizes around the cap region of the PilQ oligomer. This suggests a role for PilP in pilus biogenesis. Although PilQ does not need PilP for stabilization or membrane localization, the specific interaction between these two proteins indicates that they may have another coordinated activity in pilus extrusion and retraction or in related functions.

Reference:

• Balasingham, S. V.; Collins, R. F.; Assalkhou, R.; Homberset, H.; Frye, S. A.; Derrick, J. P., and Tonjum, T.: Interactions between the Lipoprotein PilP and the Secretin PilQ in Neisseria meningitidis. J. Bacteriol., 189, 5716-5727 (2007).
  • Abstract (courtesy of the Journal of Bacteriology).

More on sample preparation, labeling and microscopy:

• Collins, R. F.; Frye, S. A.; Balasingham, S.; Ford, R. C.; Tonjum, T., and Derrick, J. P.: Interaction with type IV pili induces structural changes in the bacterial outer membrane secretin PilQ. J. Biol. Chem., 280, 18923-18930 (2005).
  • Abstract (courtesy of the Journal of Biological Chemistry).

More information:

• Catalog information
• Product information, instructions and protocols
• NTA-Ni(II)-Nanogold: References
• Nanogold for cryo-EM: References
• Our paper from Microscopy and Microanalysis 2002
• 5nm NTA-Ni(II)-Gold: our paper from Microscopy and Microanalysis 2005

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Nanogold®, Synaptic Vesicle Membrane Retrieval and Synapse Development

Epidermal growth factor receptor pathway substrate clone 15 (Eps15) has been found in protein complexes with multiple endocytic proteins, including dynamin and the vertebrate homologue of Dap160, intersectin. This protein has been implicated in endocytosis, endosomal protein sorting, and cytoskeletal organization, but its role is still unclear for several reasons, including limitations of dominant-negative experiments, and apparent redundancy with other endocytic proteins. The authors used a combination of immunofluorescence, immunoprecipitation and electrophysiological experiments with Drosophila eps15-null mutants, and showed that Eps15 is required for proper synaptic bouton development and normal levels of synaptic vesicle (SV) endocytosis.

Pre-embedding immunogold was used to visualize the distribution of eps15 at points during endocytosis. Muscles were dissected from third instar Drosophila larvae, fixed in 4% paraformaldehyde and embedded in agarose. Vibratome slices of the agarose blocks were incubated with guinea pig anti-Eps15 antiserum, followed by Nanogold-conjugated secondary antibodies. The immunogold labeling was silver enhanced using an IntenSE Silver Enhancement kit from GE Healthcare; samples were then embedded in Durcupan ACM for ultrathin sectioning. Serial ultrathin sections were counterstained with uranyl acetate and lead citrate, then examined in a transmission electron microscope. Images were quantified using NIH ImageJ, and statistical evaluation was performed using Excel (Microsoft). It was noted that the silver enhancement procedure, which works by the precipitation of silver around the gold particles, resulting in the formation of irregularly shaped black precipitates; our HQ Silver silver enhancement reagent is formulated for the most uniform and regular particle development, and may help produce more regular enhanced particles in this application.

Consistent with a role in SV endocytosis, electron microscopic analysis and quantitation of Eps15 within cellular compartments showed that it moves from the center of synaptic boutons to the periphery in response to synaptic activity. The endocytic protein, Dap160/intersectin, is a major binding partner of Eps15; eps15 mutants phenotypically resemble dap160 mutants. Analyses of eps15 dap160 double mutants suggest that Eps15 and Dap160 play a coordinated role in SV endocytosis. The authors infer that Eps15 and Dap160 promote the efficiency of endocytosis from the plasma membrane by maintaining high concentrations of multiple endocytic proteins, including dynamin, at synapses.

Reference:

• Koh T. W.; Korolchuk V. I.; Wairkar Y. P.; Jiao W.; Evergren E.; Pan H.; Zhou Y.; Venken K. J, Shupliakov O.; Robinson I. M.; O'kane C. J., and Bellen H. J.: Eps15 and Dap160 control synaptic vesicle membrane retrieval and synapse development. J. Cell Biol., 178, 309-322 (2007).
  • Abstract (courtesy of the Journal of Cell Biology).

More information:

• Catalog information: Nanogold conjugates
• Instructions and product information: Nanogold-Fab' and IgG conjugates
• Pre-embedding immunoelectron microscopy with Nanogold and HQ Silver enhancement: application feature
• Technical help: Nanogold conjugates
• Nanogold references, sorted by application

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The Right Contacts: Ordering, Technical Questions, and Custom Synthesis

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:

More information:

• How to Order
• Custom synthesis: general information
• Custom synthesis terms and conditions
• International Distributor list
• How to order from our international distributors
• News (holiday hours, closings)

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Other Recent Publications

Quantitative analysis of the ultrastructural distribution of the granules and of the immunogold particles bound to the wtPABD-EGFP probe was performed in 21 transfected chromaffin cells after stimulation. Granules as well as gold particles were considered to be associated with cellular structures (plasma membrane, subplasmalemmal shell (SPM), and granule membrane) when separated by <50 nm. The culture and fixation conditions used in this study yielded SPM thicknesses of 184.5 ± 3.9 nm. The percentage of granule in the SPM region and non-SPM internal cytoplasm of the cells was counted to estimate the current secretory activity of the stimulated cells compared with resting cells, which did not display any granule in SPM shell. The density of immunogold particles in the SPM region and in the internal cytoplasm was measured using the image analysis software Axiovision AC Rel. 4.5; it was determined as the number of particles/µm² of area. Percentages of plasma membrane- and granule-bound immunogold particles in SPM were calculated relative to total immunogold particles counted in SPM; and finally, to estimate the particular accumulation of PA at the plasma membrane involved in granule docking, we compared the numbers of immunogold particles/µm of plasma membrane at docking sites versus non-docking sites of the plasma membrane (t test).

Phospholipase D1 was found to be activated in secretagogue-stimulated cells, and it produces PA at the plasma membrane at secretory granule docking sites. Phospholipase D1 activation and PA production were shown to represent key events in the exocytotic progression. Membrane capacitance measurements indicate that reduction of endogenous PA impairs the formation of fusion-competent granules. These findings demonstrate that PA synthesis is required during exocytosis to facilitate a late event in the granule fusion pathway, and the authors propose that the underlying mechanism is related to the ability of PA to alter membrane curvature and promote hemi-fusion.

Reference:

• Zeniou-Meyer, M.; Zabari, N.; Ashery, U.; Chasserot-Golaz, S.; Haeberle, A. M.; Demais, V.; Bailly, Y.; Gottfried, I.; Nakanishi, H.; Neiman, A. M.; Du G.; Frohman, M. A.; Bader, M. F., and Vitale, N.: Phospholipase D1 Production of Phosphatidic Acid at the Plasma Membrane Promotes Exocytosis of Large Dense-core Granules at a Late Stage. J. Biol. Chem., 282, 21746-21757 (2007).
  • Abstract (courtesy of the Journal of Biological Chemistry).

Reference for silver enhancement procedure:

• Yi, H.; Leunissen, J.; Shi, G.; Gutekunst, C., and Hersch, S.: A novel procedure for pre-embedding double immunogold-silver labeling at the ultrastructural level. J. Histochem. Cytochem., 49, 279-284 (2001).
  • Abstract (courtesy of the Journal of Histochemistry and Cytochemistry).
  • Reprint (PDF) (courtesy of the Journal of Histochemistry and Cytochemistry).

Although we have often reported the fluorescence quenching properties of Nanogold and other gold nanoparticles and their application to constructs such as molecular beacons, larger metal nanoparticles can also enhance the fluorescence of molecules positioned at specific distances from their surface. Joseph Lakowicz and group, who have pioneered this field, extend it to multiple metal particle constructs in a recent paper in Nano Letters. 20 nm silver particle dimers with single Cy5 molecules localized between coupled metal particles were prepared by hybridization of 20 nm silver particle-conjugated single-stranded oligonucleotides with double-length single-stranded oligonucleotides containing single Cy5 molecules. Image analysis revealed that the single-molecule fluorescence was enhanced 7-fold on the metal monomer and 13-fold on the metal dimer relative to the free Cy5-labeled oligonucleotide in the absence of metal. Lifetimes were shortened on the silver monomers and further shortened on the silver dimers, demonstrating the near-field interaction mechanism of fluorophore with the metal substrate. Finite-difference time-domain (FDTD) calculations were employed to study the distribution of electric field near the metal monomer and dimer, providing a method to study the coupling effect of metal particle on the fluorescence enhancement.

Reference:

• Zhang, J.; Fu, Y.; Chowdhury, M. H., and Lakowicz, J. R.: Metal-Enhanced Single-Molecule Fluorescence on Silver Particle Monomer and Dimer: Coupling Effect between Metal Particles. Nano Lett., 7, 2101-2107 (2007).
  • Abstract (courtesy of ACS Publications).

Priddy and colleagues, meanwhile, were doing correlative microscopy in reverse, using a structure previously determined by scanning transmission electron microscopy (STEM) and labeling with Nanogold®-labeled calmodulin as the basis for the design of fluorescence resonance energy transfer experiments to show the conformational substates of calmodulin bound to the phosphorylase kinase complex. As they describe in a recent Protein Science paper, they exchanged the delta subunits of the phosphorylase kinase (PhK) complex with a CaM double-mutant derivatized with a fluorescent donoracceptor pair (CaM-DA) to assess the conformational substates of PhKd by single molecule fluorescence resonance energy transfer (FRET) with and without Ca²⁺. The exchanged subunits were determined to occupy distinct conformations, depending on the absence or presence of Ca²⁺, as signaled by alterations of the compact, mid-length, and extended populations of their FRET distance distributions. The combined predominant mid-length and less common compact conformations of PhKd became less abundant in the presence of Ca²⁺, with the delta subunits assuming more extended conformations. This behavior contrasts with the compact forms commonly observed for many of CaMs Ca²⁺-dependent interactions with other proteins. Furthermore, the conformational distributions of the exchanged PhKdelta subunits were distinct from those of CaM-DA free in solution, ±Ca²⁺, as well as from exogenous CaM bound to the PhK complex as delta'. delta' is distinct from delta in that it binds only in the presence of Ca²⁺, but stoichiometrically and at a different location in the complex.

Reference:

• Priddy, T. S.; Price, E. S.; Johnson, C. K., and Carlson, G. M.: Single molecule analyses of the conformational substates of calmodulin bound to the phosphorylase kinase complex. Protein Sci., 16, 1017-1023 (2007).
  • Abstract (courtesy of Protein Science).

Original Nanogold labeling and Scanning Transmission Electron Microscopy Structure:

• Traxler, K. W.; Norcum, M. T.; Hainfeld, J. F., and Carlson, G. M.: Direct visualization of the calmodulin subunit of phosphorylase kinase via electron microscopy following subunit exchange. J. Struct. Biol., 135, 231238 (2001).
  • Abstract (courtesy of Science Direct).

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