Updated: April 10, 2002

N A N O P R O B E S     E - N E W S

Vol. 3, No. 4          April 10, 2002

This monthly newsletter is keep you informed about techniques to improve your immunogold labeling, highlight interesting articles and novel metal nanoparticle applications, and answer your questions. We hope you enjoy it and find it useful.

Have questions, or issues you would like to see addressed in the next issue? Let us know by e-mailing tech@nanoprobes.com.

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Nanogold® Electron Crystallography of the Scrapie Prion Protein

The extreme insolubility of the aberrantly folded isoform (PrPSc) of the prion protein (PrP) responsible for Creutzfeldt-Jakob disease (CJD), bovine spongiform encephalopathy (BSE) and other spongiform encephalopathies has prevented structural determination by X-ray diffraction or NMR. However, Wille, Agard and co-workers have used Monoamino Nanogold® labeling and 2-D electron crystallography of two truncated but still infectious variants, N-terminally truncated PrPSc (PrP 27-30) and a miniprion (PrPSc106), to construct models for the PrPSc structure. N-linked sugars were oxidized with periodate, then selectively labeled using Monoamino Nanogold to allow localization of the sugars. Negative stain electron microscopy and image processing allowed the extraction of limited structural information to 7 resolution. The 36-residue internal deletion of the miniprion and N-linked sugars were localized by comparing projection maps of PrP 27-30 and PrPSc106. The dimensions of the monomer and the locations of the deleted segment and sugars were then used as constraints in the construction of models for PrPSc: these were satisfied only by structures featuring parallel beta-helices as the key element, a model with implications for understanding prion propagation and the pathogenesis of neurodegeneration. Reference:

Wille, H.; Michelitsch, M. D.; 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, 99, 3563-8 (2002).

The procedure for labeling glycoproteins with Monomaleimido Nanogold using periodate oxidation of sugar residues is given in the product information for Monoamino Nanogold, available on our web site. A general procedure for labeling sugar residues in RNA using Monomaino Nanogold and periodate oxidation is also given on our web site:


More information:

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Colloidal Gold Conjugation Procedures and Applications

Looking for an effective conjugation procedure for colloidal gold? Research Diagnostics, Incorporated include a useful procedure for antibody conjugation on their web site:


Find out about the use of gold probes for lateral flow devices and other medical diagnostics, the types of gold probes used, and the factors that are important, in this article by John Chandler, Tracey Gurmin, and Nicola Robinson from IVD Technology magazine, March 2000:

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Nanovan and Nano-W: Light and 'Tunable' Negative Staining

Because Nanogold® is relatively small, it may be difficult to visualize in the presence of heavy metal based stains such as uranyl acetate or lead citrate. However, if you are negative staining - staining the regions between or around the edges of your specimens - you can use NanoVan, a lighter stain based on vanadium, which gives a highly uniform but much less electron-dense staining against which the Nanogold particles are easily visualized. Furthermore, by mixing with our tungsten-based negative stain, Nano-W, you can "tune" the opacity to suit your application.

Gregori and co-workers describe the use of NanoVan with a Nanogold-labeled peptide to determine the binding site of the peptide within the proteasome. Reference:

Gregori, L.; Hainfeld, J. F.; Simon, M. N.; and Goldgaber, D. Binding of amyloid beta protein to the 20S proteasome. J. Biol. Chem., 272, 58-62 (1997).

Reprint (pdf): http://www.jbc.org/cgi/reprint/272/1/58.pdf

Zagursky and co-workers used NanoVan in conjunction with 12 nm colloidal gold labeling to localize a surface-exposed, highly conserved, immunogenic nontypeable Haemophilus influenzae (NTHi) protein, NucA, which elicits cross-reactive bactericidal antibodies against NTHi and may form the basis for a vaccine against NTHi disease, on the sruface of NTHi cells. Reference:

Zagursky, R. J.; Ooi, P.; Jones, K. F.; Fiske, M. J.; Smith, R. P., and Green, B. A.: Identification of a Haemophilus influenzae 5'-nucleotidase protein: cloning of the nucA gene and immunogenicity and characterization of the NucA protein. Infect. Immun., 68, 2525-34 (2000).

Reprint (pdf): http://iai.asm.org/cgi/reprint/68/5/2525.pdf

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The Advantages of Gold Enhancement

GoldEnhance works like silver enhancement, but instead deposits gold. Gold Enhanced particles are denser than silver enhanced ones for superior back-scattered electron (BSE) detection in the scanning electron microscope (SEM). Gold enhancement is impervious to osmium etching, may be used in the presence of chloride buffers without precipitation problems. It is also not etched by osmium tetroxide, and provides an alternative enhancement method if you are having this problem with silver enhancement. GoldEnhance also produces excellent results for light microscopy; see our references page for details. Silver-enhanced immunogold labeling of specimens on metal substrates is problematic because the substrates promote severe osmium etching, even though smaller gold particles produce much more successful labeling. Owen and co-workers now report that GoldEnhance is not affected by osmium in this application: they successfully labeled vinculin in the focal adhesion sites of fibroblasts using 5 nm gold with gold enhancement, and observed labeling by SEM. Reference:

Owen, G. R.; Meredith, D. O.; Ap Gwynn, I., and Richards, R., G.: Enhancement of immunogold-labelled focal adhesion sites in fibroblasts cultured on metal substrates: problems and solutions. Cell. Biol. Int., 25, 1251-1259 (2001).

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Other Recent Publications

Mirkin and co-workers report a novel DNA array detection method utilizing the binding of oligonucleotides functionalized with gold nanoparticles to localize gold nanoparticles in an electrode gap; silver deposition bridges the gap and leads to readily measurable conductivity changes. Target DNA at concentrations as low as 500 femtomolar were detected with a point mutation selectivity factor of approximately 100,000:1. Reference:

Park, S. J, Taton, T. A, and Mirkin, C. A.: Array-based electrical detection of DNA with nanoparticle probes. Science, 295, 1503-6 (2002).

Abstract (Medline): http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=11859188&dopt=Abstract

Friml and co-workers describe the use of 10 nm colloidal gold from Nanoprobes (via our distributor in Germany, BioTrend) in thin sections to localize PIN3 protein, a component of the lateral auxin transport system regulating tropic growth. Reference:

Friml, J.; Wisniewska, J.; Benkova, E.; Mendgen, K., and Palme, K.: Lateral relocation of auxin efflux regulator PIN3 mediates tropism in Arabidopsis. Nature, 415, 806-9 (2002).

Abstract (Medline): http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=11845211&dopt=Abstract

Lipschitz and Michel describe the use of 1 nm and 15 nm colloidal gold particles to demonstrate the specific activation of predominantly microvillar olfactory sensory neurons (OSNs) in the zebrafish olfactory epithelium by amino acid odorants, using the ion-channel permeant probe agmatine and detecting with 1 nm gold-labeled secondary antibodies and silver enhancement forlight microscopy, and 15 nm gold-labeled antibodies for electron microscopy. However, they wrongly describe both these probes as "nanogold." Please remember - only the 1.4 nm covalently linked gold label made by Nanoprobes is called Nanogold® - and it is trademarked! Reference:

Lipschitz, D. L., and Michel, W. C.: Amino Acid Odorants Stimulate Microvillar Sensory Neurons. Chem. Senses, 27, 277-286 (2002).

Abstract (Medline): http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=11923189&dopt=Abstract

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