N A N O P R O B E S     E - N E W S

Vol. 8, No. 12          December 22, 2007

• Higher labeling resolution. The nitrilotriacetic acid - Ni(II) chelate is much smaller than an antibody or protein, and therefore when it is bound, the gold is much closer to its target. This makes NTA-Ni(II)-Nanogold ideal for localizing sites in protein complexes or other 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, and perturbs their ultrastructure less. In some systems it may be used with stronger fixation or less permeabilization, enabling labeling with better ultrastructural preservation.

• 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.

• 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 functionalization. Dissociation constants are estimated to be between 10^-7 to 10^-13 M^-1. For many applications, this provides binding strengths comparable to antibodies.

Applications include:

• High-resolution labeling of proteins, protein complexes or organelles containing recombinant His-tagged proteins for EM1 or STEM2 localization.
• Labeling and molecular localization of two different subunits of Photosystem II.
• "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.

Features and advantages include:

• Detect His-tagged proteins in an hour.
• Direct visualization of His-tagged proteins in magenta colored bands. No film, autoradiography or phosphorimager are required.
• More stable than antibodies.
• Low nanogram-level sensitivity with low background.
• No antibodies involved.

Applications include:

• Identify His-tagged proteins rapidly and confidently in cell lysates and extracts.
• Faster, simpler western blots.
• Confirm the expression of his-tagged reporter proteins in transfected cells.

Reference for Ni-NTA-Nanogold:

• Hainfeld, J. F.; Liu, W.; Halsey, C. M. R.; Freimuth, P., and Powell, R. D.: Ni-NTA-Gold Clusters Target His-Tagged Proteins. J. Struct. Biol., 127, 185-198 (1999).
  • Abstract (courtesy of Science Direct).

More information:

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

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How to Get the Best out of Ni-NTA-Nanogold®

• Wash with a buffer containing imidazole. Imidazole is the active coordinating group in histidine; treatment with imidazole will displace isolated single histidine binding, but it will not overcome the chelate effect and stronger binding of NTA with polyhistidines, and therefore will disturb polyhistidine binding much less. Try increasing the imidazole concentration from 10 mM progressively to 200 mM until background is controlled to your satisfaction.

• Increase the ionic strength of the solution. The nitrilotriacetic acid moiety is negatively charged, and may interact with positively charged regions of a target; increasing the ionic strength will help to prevent this. Try 300 mM NaCl, and, if your biomolecule can tolerate it, increase to 1.0 M if necessary.

• Other possible factors include:
  • hydrophobic interactions. These may be reduced or eliminated by the addition of detergents; try 0.05% Tween-20, and increase to 0.1% if necessary.
  • pH: it may be helpful to vary the pH to find an optimum pH at which charge interactions such as those mentioned above are reduced.
  • Transition metals may also promote interactions between the NTA group and electron donating groups in your specimen. Wash with 0.05 M disodium ethylene diamine tetraacetic acid (EDTA) to remove these.
  • Interaction of thiols (sulfhydryls) with gold. Thiols have a strong affinity for gold, and if they are present in your specimen, any gold particle species, including Ni-NTA-Nanogold, may bind to them. Avoid the use of reducing buffers or preservatives containing thiols, such as dithiothreitol (DTT), mercaptoethanol, or mercaptoethylamine hydrochloride. If your specimen contains exposed thiols, they may be blocked with N-ethylmaleimide.

If you are seeing background signal after silver enhancement or gold enhancement, we have found that including 5% nonfat dried milk in the blocking buffer to be helpful in controlling background with Nanogold reagents. Including 0.1% Tween-20 in both the blocking and incubation buffers is also helpful. A number of methods are available for controlling background, based on stopping the development reaction and preventing further reaction after the desired end-point by reagents that have diffused into specimens.

In the development of Ni-NTA-Nanogold, it was found that a form containing multiple NTA-Ni(II) groups produced the best overall combination of labeling selectivity, density and sensitivity. However, because this can interact with polyhistidine tags on several protein molecules simultaneously, it may act to aggregate proteins, or perturb the formation of protein complexes, in solution. You can avoid this by using a ratio of reagent to protein such that the stoichiometry reduces or eliminates this possibility. For example, if your protein has only one polyhistidine tag, then using an excess of the Ni-NTA-Nanogold® reagent will guard against the possibility of multiple interactions. You can also help avoid the possibility by carefully selecting when to add the reagent, for example after complex assembly.

More information:

• NTA-Ni(II)-Nanogold: catalog information
• NTA-Ni(II)-Nanogold: product information and instructions
• Technical help for NTA-Ni(II)-Nanogold
• NTA-Ni(II)-Nanogold: References
• 5 nm Ni-NTA-Gold: our paper from Microscopy and Microanalysis 2005

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Monoamino Nanogold®: Localization of Sugars in the Scrapie Prion Protein

Applications of Monoamino Nanogold:

• Labeling glycoproteins for microscopic observation and localization.
• Label tRNA for electron microscopic localization.
• Activate the phosphate group in phospholipids or oligonucleotides then label with Monoamino Nanogold.

In their recent paper in the Archives of Biochemistry and Biophysics, Holger Wille and group described the structure of the disease-causing isoform of the prion protein (PrP^Sc) using electron crystallography with heavy atom negative staining. Previously, the insolubility of this isoform (PrP^Sc) has prevented studies of its three-dimensional structure at atomic resolution. This study followed up on an earlier paper in which the authors had used Monoamino Nanogold labeling of oxidized sugar residues to localize the N-linked oligosaccharides of PrP 2730, reflecting the differences in glycosylation between PrP 2730 and PrP^Sc 106; this illustrates both the role of heavy atom labels such as Nanogold in structural studies, and the application of multiple methods to arrive at full structures for difficult entities such as the prion isoform.

The labeling procedure used for the sugar residues was based on that previously used with undecagold. Suspensions of PrP 27-30 2D crystals were oxidized with 10 mM NaIO₄ in 100 mM HEPES-sodium hydroxide (pH 7.0) for 2 hours at room temperature (RT): the protein was kept in suspension by end-over-end rotating. After the oxidation, the protein was pelleted and resuspended in 100 mM sodium carbonate buffer, pH 9.0. An excess of Monoamino Nanogold was suspended in carbonate buffer and added to the protein. The oxidized 2D crystals and the Monoamino Nanogold were reacted for 2 hours at RT under constant rotation. The reaction was quenched by adding a small aliquot of 5 M sodium borohydride in 0.1 M NaOH. The reaction mixture was then allowed to sit undisturbed for 30 minutes at RT. Finally, the protein was pelleted, the unbound gold label discarded with the supernatant; the pellet was resuspended in buffer. Controls were treated identically with the exception that no Monoamino Nanogold was added.

The sugar residues within the glycosylphosphatidylinositol (GPI) anchor made it necessary to establish that the label was selectively bound to the N-linked sugars. To test, gold-labeled PrP 27-30 was denatured and treated with PNGase F. Western blots of unlabeled, labeled, and enzyme-treated samples were developed with the 3F4 Ab or by silver enhancement to directly visualize the gold label on the blotting membrane. The labeled samples showed an additional band at ~45 kDa that was detected by both 3F4 and the silver enhancement, indicating that this band corresponded to the gold-labeled PrP 27-30. Treatment with PNGase F removed the gold label, thus showing that the Nanogold label indeed specifically bound to the N-linked sugars.

In the current experiment, PrP 27-30 and PrP^Sc 106 were negatively stained with heavy metals, and the heavy atom deposits localized by electron crystallography. The interactions of the heavy metals with the crystal lattice were governed by tertiary and quaternary structural elements of the protein, as well as the charge and size of the heavy metal salts. Staining with molybdate anions revealed three prominent densities near the center of the trimer that forms the unit cell, coinciding with the location of the beta-helix that was proposed for the structure of PrP(Sc). Differential staining also confirmed the location of the internal deletion of PrP(Sc)106 at or near these densities.

Reference:

• Wille H.; Govaerts C.; Borovinskiy A.; Latawiec D.; Downing K. H.; Cohen F. E., and Prusiner S. B.: Electron crystallography of the scrapie prion protein complexed with heavy metals. Arch. Biochem. Biophys., 467, 239-248 (2007)
  • Abstract (courtesy of Science Direct).

Reference for Monoamino Nanogold labeling:

• Wille, H.; Michelitsch, MD.; Guenebaut, V.; Supattapone, S.; Serban, A.; Cohen, F. E.; Agard, D. A, and Prusiner, S. B.: Structural studies of the scrapie prion protein by electron crystallography. Proc. Natl. Acad. Sci. USA., , 3563-3568. (2002)
  • Abstract (courtesy of the National Academy of Sciences of the USA).
  • Reprint (PDF) (courtesy of the National Academy of Sciences of the USA).

More information:

• Monoamino Nanogold: catalog information
• Monoamino Nanogold: product information and instructions
• Technical help: Nanogold labeling reagents
• Monoamino Nanogold: References

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Pre-embedding Immunogold Labeling: Best Procedures with Nanogold®

In a recent paper in Neuroscience, Tao-Cheng describes the use of Nanogold pre-embedding labeling for the ultrastructural localization of active zone and synaptic vesicle proteins in a preassembled multi-vesicle transport aggregate. Although it has been suggested that the presynaptic active zone (AZ) may be preassembled, it is still unclear which entities carry the various proteins to the AZ during synaptogenesis, and Tao-Cheng uses Nanogold labeling of component proteins to investigate her hypothesis that aggregates of dense core vesicles (DCV) and small clear vesicles in the axons of young rat hippocampal cultures are carriers containing preformed AZ and synaptic vesicle (SV) components on their way to developing synapses.

Dissociated hippocampal cells from 21-day embryonic SpragueDawley rats were fixed using a variety of fixatives, each selected for optimal immunolabeling with the different antibodies used: (1) 4% paraformaldehyde in phosphate-buffered saline (PBS) for 4560 minutes for all antibodies; (2) 4% paraformaldehyde and 0.020.1% glutaraldehyde for 3060 minutes for the SV2 antibody, and for 3045 minutes for the guinea-pig Piccolo antibody; (3) 2% acrolein in PBS for 1 minute followed by 4% paraformaldehyde in PBS for 3060 minutes for the guinea-pig Piccolo and the SV2 antibodies; and (4) 4% glutaraldehyde in 0.1 M sodium cacodylate buffer for 1 hour for samples without immunolabeling. Fixed cells were washed and permeabilized/blocked with 0.1% saponin/5% normal goat serum in phosphate-buffered saline (PBS) for 1 hour, incubated with primary antibody for 12 hours, then incubated with the Nanogold-labeled secondary antibody for 1 hour, and then silver enhanced using HQ silver for 1015 minutes, treated with 0.2% OsO₄ in phosphate buffer for 30 minutes. Samples were then stained with 0.25% uranyl acetate at 4°C overnight, dehydrated in ethanol, and embedded in epoxy resin. Controls for specificity of immunolabeling included omitting the primary antibody, and using the different primary antibodies as controls for each other.

The aggregates were positively labeled with antibodies against Bassoon and Piccolo (two AZ cytomatrix proteins), VAMP, SV2, synaptotagmin (three SV membrane proteins), and synapsin I (a SV-associated protein). Bassoon and Piccolo labeling were localized at dense material both in the aggregates and at the AZ. In addition to the SV at the synapses, antibodies to the SV membrane proteins also labeled the clear vesicles in the aggregate, as well as many other SV-like and pleiomorphic vesicular structures in the axons. Synapsin I labeling was also associated with the vesicles in the aggregates. In single sections, these axonal vesicle aggregates were ~0.22 by 0.13 µm in average dimensions, and were found to contain one to two DCV and five to six small clear vesicles. Labeling in serial sections confirmed that the aggregates were not synaptic junctions sectioned en face. Labeling intensities of Bassoon and Piccolo measured from serially sectioned transport aggregates and AZ were within range of each other, suggesting that one or a few aggregates, but not individual DCV, can carry sufficient Bassoon and Piccolo to form an AZ. The present findings provide the first ultrastructural evidence localizing various AZ and SV proteins in a preassembled multi-vesicle transport aggregate that has the potential to quickly form a functional active zone.

Reference:

• Tao-Cheng, J.-H.: Ultrastructural localization of active zone and synaptic vesicle proteins in a preassembled multi-vesicle transport aggregate. Neuroscience, 150, 575-584 (2007).
  • Abstract (courtesy of Science Direct).

More information:

• Special Feature: Nanogold pre-embedding immunoelectron microscopy with HQ Silver enhancement
• Nanogold conjugates: catalog information
• Nanogold conjugates: product information and instructions
• References: Nanogold for pre-embedding immunolabeling
• Technical help: Nanogold conjugates

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Nanoprobes Holiday Hours

• Contact information
• Nanoprobes: news and press releases
• About Us: company information

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Other Recent Publications

Reference:

• Giljohann, D. A.; Seferos, D. S.; Patel, P. C.; Millstone, J. E.; Rosi, N. L.; and Mirkin, C. A.: Oligonucleotide Loading Determines Cellular Uptake of DNA-Modified Gold Nanoparticles. Nano Lett., 7, 3818-3821 (2007).
  • Abstract (courtesy of ACS Publications).

There's even more on transfection: In a recent article in Biomaterials, Zhou and group report on the advantages of gold nanoparticles in gene delivery systems based on conventional high molecular weight chitosans. These molecules are efficient for DNA vaccine delivery, but have poor physical properties including aggregation, low solubility at neutral pH, high viscosity at concentrations used for in vivo delivery, and a slow onset of action. Chitosan oligomers shorter than 14 monomers units have been found to form only weak, unstable complexes with DNA, resulting in physically unstable polyplexes in vitro and in vivo. However, low molecular weight chitosans with an average molecular mass of 6 kDa (Chito6) were attached to gold nanoparticles (GNPs) by preparing gold nanoparticles using reduction of tetrachloroaurate in the presence of Chito6. The potency of the resulting Chito6-GNPs conjugates as vectors for the delivery of plasmid DNA was investigated in vitro and in vivo. After delivery by intramuscular immunization in BALB/c mice, the Chito6-GNP conjugates induced an enhanced serum antibody response 10 times more potent than naked DNA vaccine. In contrast to naked DNA, the Chito6-GNPs conjugates also induced potent cytotoxic T-lymphocyte responses at a low dose.

Reference:

• Zhou, X.; Zhang, X.; Yu, X.; Zha, X.; Fu, Q.; Liu, B.; Wang, X.; Chen, Y.; Chen, Y.; Shan, Y.; Jin, Y.; Wu, Y.; Liu, J.; Kong, W., and Shen, J.: The effect of conjugation to gold nanoparticles on the ability of low molecular weight chitosan to transfer DNA vaccine. Biomaterials, 29, 111-117 (2008).
  • Abstract (courtesy of Science Direct).

Tao-Cheng is not the only researcher using Nanogold for pre-embedding immunolabeling. In a recent paper in the Journal of Cell Biology, Graser, Stierhof and co-workers use this method to localize centrosomal proteins in primary cilia (PC) formation. Primary cilia (PC) function as microtubule-based sensory antennae projecting from the surface of many eukaryotic cells. They play important roles in mechano- and chemosensory perception, and their dysfunction is implicated in developmental disorders and severe diseases. The basal body that functions in PC assembly is derived from the mature centriole, a component of the centrosome. One newly identified protein, Cep164, was found to be indispensable for PC formation, and therefore was characterized in detail using pre-embedding Nanogold labeling. For preembedding immuno-EM, U2OS cells were grown on coverslips, fixed with 4% formaldehyde for 10 minutes, and permeabilized with phosphate-buffered saline (PBS) with 0.5% Triton X-100 for 2 minutes. Cells were blocked with 1% bovine serum albumin (BSA) in PBS for 10 minutes, washed three times in PBS, then incubated with primary antibodies: these were 1 µg/mL affinity-purified rabbit anti-Cep164 IgG (R171) and antiC-Nap1 IgG; mouse anti-Cep170 mAb (77-419-2); antigamma-tubulin mAb (1:1,000, GTU-88), anti-centrin mAb (1:3,000, 20H5), and anti-acetylated tubulin mAb (1:2,000; 6-11B-1). This was followed by incubation with goat antirabbit IgG-Nanogold; the Nanogold was then silver enhanced with HQ Silver. Cep164 was localized to the distal appendages of mature centrioles. In contrast to ninein and Cep170, two components of subdistal appendages, Cep164 persisted at centrioles throughout mitosis. Moreover, the localizations of Cep164 and ninein/Cep170 were mutually independent during interphase. These results implicate distal appendages in PC formation, and identify Cep164 as an excellent marker for these structures.

Reference:

• Graser, S.; Stierhof, Y.-D.; Lavoie, S. B.; Gassner, O. S.; Lamla, S.; Le Clech, M., and Nigg, E. A.: Cep164, a novel centriole appendage protein required for primary cilium formation. J. Cell Biol., 179, 321-330 (2007).
  • Abstract (courtesy of the Journal of Cell Biology).

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