N A N O P R O B E S     E - N E W S

Vol. 9, No. 1          January 31, 2008

We have now found that EnzMet is one of the cleanest and most sensitive detection reagents we have tried for Western blots. A comparison between conventional DAB development and EnzMet development on a Western blot is shown below:

In most cases, the EnzMet protocol using EnzMet for Blots below may be substituted for conventional DAB development without further modification of the protocol. However, because of its greater sensitivity, greater dilutions of either primary antibody or secondary probes may be required to achieve the optimum combination of sensitivity and clarity. A five-fold to ten-fold additional dilution has been found to give good results in immunohistochemical experiments and is likely to be appropriate here also.

Protocol:

1. Wash with buffer containing 0.1% Tween-20 for 3 x 5 minutes.
   Note: Phosphate buffered saline, tris buffered saline or other wash buffers can be used. Including 0.1 % (w/v) Tween-20 in the wash buffer was found to be helpful in reducing non-specific binding.

2. Wash with deionized water for 3 x 5 minutes.

3. Shake off excess water. Cover membrane with 6 mL (or 3 volumes) of EnzMet Detect A. Incubate for 4 minutes.
   Note: Excess water can lead to the dilution of EnzMet reagents, resulting in weak staining and results which are difficult of reproducing.

4. Add 2 mL (or 1 volume) of EnzMet Detect B to the membrane, and gently mix Solutions A and B. Incubate for 4 minutes.

5. Add 2 mL (or 1 volume) of EnzMet Detect C to the membrane, and gently mix Solutions A, B and C. Incubate for 9 - 25 minutes, or until satisfactory staining is achieved.
   Note: The EnzMet incubation time mainly depends on the target concentrations and staining temperature. Longer incubation may be needed for visualizing low concentration targets. However, longer incubation may lead to some non specific background staining. The variation of EnzMet staining temperature can affect its silver deposition rate. Lower temperature slows down the deposition process, and thus a longer staining time may be required to reach a certain degree of staining density and sensitivity.

6. Wash with deionized water for 3 x 5 minutes.

7. Air dry membrane for record.

In addition to its use for staining and blotting, EnzMet may also be used for highly specific electrical detection using conductive array biochips: enzyme-labeled DNA probes were developed with EnzMet to form conductive bridges after binding to targets patterned to connect two electrodes.

References:

• 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).
• Tubbs R.; Pettay J.; Powell R.; Hicks D. G.; Roche P.; Powell W.; Grogan T., and Hainfeld, J. F.: High-resolution immunophenotyping of subcellular compartments in tissue microarrays by enzyme metallography. Appl. Immunohistochem. Mol. Morphol., 13, 371-375 (2005).
  • Abstract (courtesy of the PubMed (Medline)).
• Moller, R.; Powell, R. D.; Hainfeld, J. F., and Fritzsche, W.: Enzymatic control of metal deposition as key step for a low-background electrical detection for DNA chips. Nano Lett., 5, 1475-1482 (2005).
  • Abstract (courtesy of PubMed (Medline)).

More information:

• EnzMet: catalog and general information
• EnzMet for Blots: Product information and instructions
• EnzMet ISH / IHC: Product information and instructions
• EnzMet Applications
• Enzyme Metallography: A Simple New Staining Method (our paper from Microscopy & Microanalysis 2002

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Improved Labeling with Mono-Sulfo-NHS-Nanogold®

Although we recommend using Monomaleimido Nanogold® to label at a hinge thiol site, we have found that antibody IgG labeled using Mono-Sulfo-NHS-Nanogold gives labeling that is just as sensitive and specific. In many situations, hinge thiol labeling is not possible, and in these cases we recommend Mono-Sulfo-NHS-Nanogold: this reacts with primary amines, and every protein contains at least one primary amine which may usually be labeled directly with Mono-Sulfo-NHS-Nanogold without needing any prior preparation, such as hinge disulfide reduction:

• If you are working with small quantities of antibody and cannot afford more than one reaction step.

• If you are labeling a conjugate or other construct that is sensitive to thiols or to reducing conditions.

• If the hinge disulfides in your antibody are not amenable to reduction. In some cases reduction will not be as selective towards the hinge thiols as desired; in others, reduction results may be variable or reduction may be incomplete, leading to low labeling.

• If you require a probe that is smaller than a whole IgG molecule, but your antibody is of a class that cannot be digested with pepsin or ficin to prepare Fab' fragments to label with Monomaleimido Nanogold. In this case you can use papain to prepare Fab fragments, which you can then label with Mono-Sulfo-NHS-Nanogold.

To check whether you can prepare Fab' fragments: Pepsin will only digest IgG1 and IgG2a, while ficin produces F(ab')₂ only from IgG1. If you have an IgG2b or IgG3 antibody, this digestion will not work and you should prepare Fab fragments or use the whole IgG.

Recently, we have explored the conditions used for this reaction to determine which produce the highest and most consistent labeling, and as a result we can offer more informed and accurate suggestions to improve labeling. If you find low labeling with this reaction, we recommend that you try the following:

• We do not recommend high pH for this reaction (higher than 7.5). At higher pH values, such as pH 8.2, although amine reactivity is enhanced, the rate of hydrolysis of the Sulfo-NHS ester is also increased significantly. The half-life of the Sulfo-NHS ester at pH 8.2 and 4°C is only about 10 minutes. Because the antibody or protein to be labeled is a large macromolecule, the time required for the Nanogold and protein to assume the correct geometry for reaction is frequently longer than this, and at higher pH values, a significant proportion of the Sulfo-NHS esters may hydrolyze and lose reactivity before they have an opportunity to react with the amines on proteins. Differences in the speed with which you can reconstitute the Nanogold reagent and mix with your proteins can then make it difficult to achieve reproducibility. Therefore, relatively mild reaction conditions (pH 7.4 -7.5) are recommended for consistent labeling.

• Dissolve the Mono-Sulfo-NHS-Nanogold in deionized water, NOT buffer solution. The correct amount of buffer salt required to achieve a working buffer concentration at the appropriate pH is already included with the lyophilized Nanogold reagent.

• The appropriate procedures for monoclonal and polyclonal antibodies may differ. The reaction condition given in the product information and instructions is optimized for most polyclonal antibodies. However, for mouse monoclonal antibodies, the following conditions are recommended (note: these apply to Mono-Sulfo-NHS-Nanogold in 5 x 6 nmol aliquots; for Mono-Sulfo-NHS-Nanogold in one 30 nmol aliquot, multiply the quantities by 5):
  1. Dissolve 0.36 mg (2.4 nmol) of the monoclonal IgG in 60 µL of phosphate-buffered saline (PBS) at pH 7.5.
  2. Dissolve 2 x 6 nmol vial of Mono-Sulfo-NHS-Nanogold in 200 µL of deionized water. Add the 2 x 6 nmol of Mono-Sulfo-NHS-Nanogold to the IgG solution.
  3. Gently agitate the reaction mixture at room temperature for 1 hour.
  4. For best results, use a Millipore / Amicon YM-30 centrifuge filter to reduce the volume of the reaction mixture to reduce the volume to 0.2 mL.
  5. Do not filter the concentrated reaction mixture through a 0.2 µm filter. Inject the entire 0.2 mL reaction mixture directly onto a 16 mL Superose-12 (d = 6.6 mm, l = 50 cm) column for purification.
  6. Elute the 16 mL Superose-12 column with 0.02 M sodium phosphate buffer containing 0.15 M sodium chloride PBS pH7.4 at 12 mL/h with a fraction size of 2.5 minutes. Fractions 17-19 usually contain the mono-Nanogold-labeled IgG, and should be pooled.

More information:

• Mono-Sulfo-NHS-Nanogold: catalog information
• Mono-Sulfo-NHS-Nanogold: product information and instructions
• Technical help for Nanogold Labeling Reagents
• Separation strategies for Nanogold conjugates
• Nanogold labeling: References sorted by application

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Ni-NTA-Nanogold® and the Structure of the gamma-Tubulin Small Complex

The yeast gamma-TuSC components were expressed in Sf9 cells. The three gamma-TuSC components, Tub4p, Spc97p, and Spc98p, were coexpressed with glutathione transferase (GST)-Spc110p1220, which binds gamma-TuSC and allowed purification by glutathione affinity. GST-Spc1101220 was then separated from gamma-TuSC by anion exchange on a MonoQ column. Purified gamma -TuSC was dialyzed into HB250 (40 mM HEPES, pH 7.5, 1 mM MgCl₂, 1 mM EGTA, 1 mM dithiothreitol, 100 µM guanosine diphosphate [GDP], and 100 mM KCl), concentrated to 15 mg/mL, and 10% glycerol was added before freezing and storage at -80°C. A baculovirus construct of Tub4p with an N-terminal 6xHis tag and tobacco etch virus cleavage site was also generated. His-tagged gamma-TuSC was expressed and purified in the same manner as the untagged complex.

gamma-TuSC with an N-terminal 6xHis tag on Tub4p was labeled with nickel (II) nitrilotriacetic acid (Ni²⁺-NTA) Nanogold. Then, 100 nM gamma-TuSC was incubated in HB250 plus 250 nM gold label overnight at 4°C. Samples were prepared for EM on Quantifoil S7/2 copper grids coated with 30- to 40-Å carbon film. The grids were glow-discharged, and 4 µL of 50100 nM gamma-TuSC was applied. After 30 seconds, excess sample was wicked away, the grids were washed three times by touching to a water droplet, and stained three times by touching to a droplet of 0.75% uranyl acetate. Excess stain was removed by vacuum aspiration, and the samples left to air dry. Pairs of tilted and untilted micrographs were acquired using the conical tilt software of the UCSF tomography package on a Tecnai T20 electron microscope operating at 120 kV. Images of the sample tilted at 60° were acquired first, followed by an overlapping montage of 0° images, with a cumulative dose of 50100 e^-/Ų. The micrographs were recorded on a Gatan 4k x 4k (Gatan, Inc., Pleasanton, CA) camera at 50,000 x magnification, with a pixel size of 2.2 Å. Both untilted and tilted micrographs were taken 1.2-µm defocus to enhance the contrast between gold and the uranyl formate stain. Particles were boxed out with the EMAN program boxer and classified in SPIDER by reference-free alignment followed by PCA and hierarchical clustering to separate labeled from unlabeled particles.

gamma-TuSC is Y-shaped, with an elongated body connected to two arms. Ni-NTA-Nanogold labeling was readily apparent at the ends of the two arms, and showed that the two gamma-tubulins (Tub4) are located in lobes at the ends of the arms. The relative orientations of the other gamma-TuSC components were determined by in vivo fluorescence resonance energy transfer (FRET). The structures of different subpopulations of gamma-TuSC indicate that the connection between a mobile arm and the rest of the complex is flexible: this results in variation in the relative positions and orientations of the gamma-tubulins. In all of the structures, the gamma-tubulins are distinctly separated. This configuration is incompatible with the microtubule lattice, and the separation of the gamma-tubulins in isolated gamma-TuSC likely plays a role in suppressing its intrinsic microtubule-nucleating activity, which is relatively weak until the gamma-TuSC is incorporated into higher order complexes or localized to microtubule-organizing centers. The authors propose that further movement of the mobile arm is required to bring the gamma-tubulins together in microtubule-like interactions, and provide a template for microtubule growth.

Reference:

• Kollman, J. M.; Zelter, A.; Muller, E. G.; Fox, B.; Rice, L. M.; Davis, T. N., and Agard, D. A.: The Structure of the gamma-Tubulin Small Complex: Implications of Its Architecture and Flexibility for Microtubule Nucleation. Mol. Biol. Cell, 19, 207-215 (2008).
  • Abstract (courtesy of Molecular Biology of the Cell).

More information:

• Ni-NTA-Nanogold: Catalog information
• Product information, instructions and protocols
• Ni-NTA-Nanogold: technical help
• Ni-NTA-Nanogold: References
• Our paper from Microscopy and Microanalysis 2002

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Towards Larger Gold Labeling

Previously, we described the preparation and use of a covalent 10 nm gold immunoprobe in which 10 nm gold particles, functionalized with novel alkanethiol ligands, were cross-linked to Fab' fragments via peripheral maleimide groups in the same manner as Nanogold. These probes were compared with conventional colloidal gold probes both for immunoelectron microscopy, and for blotting:

As can be seen, both sensitivity and background cleanness are improved with the covalently linked probe. More recently, we presented some results from the preparation of a 3 nm gold-antibody conjugate, and methods for separating and identifying conjugates from unlabeled antibody and unbound gold label.

Larger versions of Ni-NTA-Nanogold® are another area of development. Since these are targeted by a metal chelate complex rather than a big targeting protein, there's room to make the gold bigger - and there are plenty of applications for a larger NTA-Ni(II) gold probe. Insertion of His tags is now feasible for a wide range of different proteins and constructs, so larger gold targeted to His tags would be a simple, versatile and effective labeling and detection reagent. At Microscopy and Microanalysis 2005, we presented preliminary results describing the preparation of a nitrilotriacetic acid - nickel (II) - 5 nm gold probe. The extended abstract of this presentation is now available on our web site.

The NTA-Ni(II)-derivatized particles were prepared similarly to the NTA-Ni(II)-Nanogold particles described previously. A transmission electron micrograph is shown below, together with a chromatogram showing the formation of a new peak corresponding to the gold conjugate when the functionalized gold particles were incubated with the protein ISWI from the ACF chromatin remodeling complex, synthesized with a His tag. In a control experiment in which the NTA-Ni(II)-derivatized particles were incubated with ISWI without the 6x-His tag, virtually no binding to the protein was seen, as evidenced by the absence of the conjugate peak.

References:

• Gutierrez, E.; Powell, R. D.; Hainfeld, J. F., and Takvorian, P. M.: A covalently linked 10 nm gold immunoprobe. Microsc. Microanal., 5, (Suppl. 2: Proceedings); G. W. Bailey, W. G. Jerome, S. McKernan, J. F. Mansfield, and R. L. Price (Eds.); Springer-Verlag, New York, NY; p. 1324-1325 (1999).
  • Complete paper (HTML format).

• Joshi, V. N.; Bhatnagar, A.; Powell, R. D., and Hainfeld, J. F.: Towards Bigger Nanogold: Preparation of Covalent 3nm Gold Fab Probes. 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, 1176CD. (2005).
  • Complete paper (HTML format).

• Reddy, V.; Lymar, E.; Hu, M., and Hainfeld, J. F.: 5 nm Gold-Ni-NTA binds His Tags. Microsc. Microanal., 11 (Suppl. 2),; 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, 1118CD (2005).
  • Complete paper (HTML format).

Keep an eye on our web site and look out for the introduction of similar reagents. In the meantime, there are options for larger gold labeling:

• If the molecule you wish to label has a cysteine or other thiol, try coordinating it directly with conventional colloidal gold; mix the molecule of interest with uncoated colloidal gold, then stabilize the resulting complex with BSA, PEG, or another protective agent.

• If you are attempting to conjugate a small molecule such as a peptide, conjugate your peptide to a macromolecule such as bovine serum albumin (BSA) or a functionalized PEG. Both of these molecules effectively stabilize colloidal gold: they bind strongly colloidal gold. You can prepare a peptide-BSA conjugate using freshly prepared 5 nm colloidal gold, then cross-link the peptide to the BSA using a heterobifunctional cross-linker such as Sulfo-SMCC (Sulfo-succinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate).

• Enlarge a Nanogold label to the appropriate size using either silver enhancement or gold enhancement. We offer two silver enhancement reagents which may then be used to enlarge our smaller 1.4 nm Nanogold reagents: HQ Silver, which is formulated for the most uniform particle size and best ultrastructural preservation in EM, and LI Silver, which is light insensitive and best for blots and light microscopy. We also offer unique gold-based autometallography reagents which deposit gold instead of silver. Either type of reagent may be used to enlarge our 1.4 nm Nanogold particles to 10 m or larger.

More information:

• A covalent 10 nm gold immunoprobe: Our paper from Microscopy and Microanalysis 1999
• Towards Bigger Nanogold:: Our paper from Microsopy and Microanalysis 2005
• 5 nm Gold-Ni-NTA binds His Tags: Our paper from Microscopy and Microanalysis 2005
• Guide to Gold Cluster Labeling: separation of conjugates
• Other product and research applications

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New Guidelines for Custom Syntheses and Contract Research

• Tetrairidium labeling reagent:
  A very small cluster, containing four iridium atoms, has been prepared as a maleimide and used to derivatize virus subunits for localization during cryo-EM and image reconstruction, and as a label for infrared microspectroscopy.

• Nanogold® and Texas Red-labeled dextran
  A 10,000 MW amino-functionalized Texas Red-labeled dextran was conjugated with Nanogold and used as a neuronal tracer.

Guidelines

Before requesting a custom synthesis quotation, we recommend that you consider our Nanogold or undecagold labeling reagents, which you may use to label a wide variety of molecules. See p. xx for more details. We are glad to advise on the preparation of gold conjugates with other molecules that are not described in this catalog: you can contact us by telephone at 1-877-447-6266 (US & Canada) or ++(01-631) 205-9490 (others), or by e-mail at tech¤nanoprobes.com.

Please note that our researchers may not be familiar with your probe or application. We can usually respond more quickly if you tell us about the molecule you wish to label:

• Molecular weight: labeling reactions use specific ratios of gold to probe. Molecular weight is used to calculate how much reagent to use, and how to separate the product.

• Optical density or UV/visible absorbtion: Extinction coefficients are known for Nanogold and undecagold. They are used to monitor the reaction and to calculate the labeling efficiency.

• References for labeling with other tags: Successful labeling experiments or protocols using enzymes or fluorophores are often a good guide to what will work with our probes and help us avoid potential problems.

• Molecular structure and functional groups: gold labeling is usually more successful when the gold is attached at a unique site located away from the binding region, so that it does not perturb the activity of your probe. We use the structure to identify the best sites and reagents for labeling.

• Solubility: The solubility of some conjugates can differ significantly from that of either the conjugate biomolecule or the gold cluster. Information on solubility and tolerance for organic solvents such as DMSO or isopropanol can help avoid or minimize reagent loss and solubility testing.

Terms and Conditions

Custom syntheses and contract research projects and products prepared by such projects are non-returnable and non-refundable. Products are intended for laboratory research purposes and may not be used for other purposes, including but not limited to human clinical trials or commercial purposes, specifically the resale of our Products to unaffiliated third parties without written approval from Nanoprobes.

Nanoprobes reserves all of its rights under any patents or other intellectual property rights covering the use, modification, or combination with other materials of its Products, technology or know-how. No rights, title or interest in any new products or materials developed during a custom synthesis or contract research project are granted or implied to the buyer by purchasing a product prepared as a custom synthesis, or entering into a contract for a research project.

Larger or more speculative projects in which significant patented or proprietary material, knowledge, expertise or know-how is supplied by both parties may require a separate agreement specifying disposition of rights to any discoveries, and discussion of such projects may require entering into a confidentiality agreement beforehand.

Nanoprobes has not tested any products for safety and efficacy in foods, drugs, biologics, medical devices, in vitro diagnostics, cosmetics or other clinical or human uses, unless expressly stated in our literature furnished with products.

More Information

For more information or to discuss a specific project, contact us by telephone at 1-877-447-6266 (US & Canada) or ++(01-631) 205-9490 (others), or by e-mail at tech@nanoprobes.com. Request a quotation online with our custom synthesis request form.

More information:

• Custom synthesis quotation request form
• Contact information
• Nanoprobes: news and press releases
• About Us: company information

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Other Recent Publications

Reference:

• Fischler, M.; Sologubenko, A.; Mayer, J.; Clever, G.; Burley, G.; Gierlich, J.; Carell, T., and Simon, U.: Chain-like assembly of gold nanoparticles on artificial DNA templates via 'click chemistry'. JCS Chem. Commun. (Camb)., 169-171 (2008).
  • Abstract (courtesy of the Royal Society of Chemistry).

While gold nanoparticles might not be the health tonic claimed by some unscrupulous merchants, they can still have therapeutic effects, as Podsiadlo and colleagues demonstrate in their recent Langmuir paper. 6-Mercaptopurine and its riboside derivatives are some of the most widely utilized anti-leukemic and anti-inflammatory drugs, but their use is restricted by their short biological half-life and severe side effects. A new delivery method for these drugs, based on conjugation to 4-5 nm gold nanoparticles, can potentially resolve these issues. The authors found substantial enhancement of the antiproliferative effect against K-562 leukemia cells of 6-mercaptopurine-9-beta-Dribofuranoside conjugated to gold nanoparticles compared to the same drug in its typically administered free form. The improvement was attributed to enhanced intracellular transport followed by the subsequent release in lysosomes. Enhanced activity and nanoparticle carriers will make possible the reduction of the overall concentration of the drug, renal clearance, and, thus, side effects. The nanoparticles with mercaptopurine also showed excellent stability over 1 year without loss of inhibitory activity.

Reference:

• Podsiadlo, P.; Sinani, V. A.; Bahng, J. H.; Kam, N. W.; Lee, J., and Kotov, N. A.: Gold nanoparticles enhance the anti-leukemia action of a 6-mercaptopurine chemotherapeutic agent. Langmuir, 24, 568-574 (2008).
  • Abstract (courtesy of the ACS Publications).

In other nanoparticle news, Figuerola and group report in a recent issue of the Journal of the American Chemical Society the one-pot preparation of size-controlled bimagnetic Iron-Platinum-Iron Oxide heterodimer nanocrystals. The authors describe a one-pot, two-step colloidal strategy to prepare bimagnetic hybrid nanocrystals (HNCs), comprising size-tuned fcc FePt and inverse spinel cubic iron oxide domains epitaxially arranged in a heterodimer configuration. The HNCs have been synthesized in a unique surfactant environment by temperature-driven sequential reactions. First, FePt NCs were prepared with sizes ranging from 3 to 9 nm by the high-temperature reduction of a platinum (II) salt with Fe(CO)₅ in an 1-octadecene solution containing oleic acid and oleyl amine surfactants at 170-200°C. FePt-iron oxide heterodimer HNCs were then prepared by further heating the crude reaction mixture containing the FePt NCs to 295°C (under N₂) at a rate of approximately 8°C/minute. The mixture was kept at this temperature for an additional 1 hour: under these circumstances, thermal decomposition of excess iron oleate complexes in the solution led to the formation of iron oxide. After this period, the flask was allowed to cool to room temperature, then opened to air. The HNCs were precipitated upon adding 2-propanol, separated by centrifugation, and finally redissolved in either hexane, toluene, or chloroform. This self-regulated mechanism offers high versatility in the control of the geometric features of the resulting heterostructures, circumventing the use of more elaborate seeded growth techniques. It has been found that, as a consequence of the exchange coupling between the two materials, the HNCs exhibit tunable single-phase-like magnetic behavior, distinct from that of their individual components and dependent upon the relative sizes of the component domains. These properties suggest that such heterodimers may be effective contrast agents for magnetic resonance imaging techniques.

Reference:

• Figuerola A.; Fiore A.; Di Corato R.; Falqui A.; Giannini C.; Micotti E.; Lascialfari A.; Corti M.; Cingolani R.; Pellegrino T.; Cozzoli PD., and Manna L.: One-pot synthesis and characterization of size-controlled bimagnetic FePt-iron oxide heterodimer nanocrystals. J. Amer. Chem. Soc., 130, 1477-1487 (2008).
  • Abstract (courtesy of the ACS Publications).

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