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Updated: December 9, 2002

N A N O P R O B E S     E - N E W S

Vol. 3, No. 12          December 9, 2002


This monthly newsletter is to 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 [email protected].

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Nanogold® as a Building Block for Nanotechnology

Kiehl and co-workers have described the self-assembly of metallic nanoparticle arrays using DNA crystals, labeled site-specifically with Nanogold®, as a programmable molecular scaffolding; this represents a critical step toward the realization of DNA nanotechnology and its nanoelectronic applications. 2-D arrays were constructed by tiling together rigid DNA motifs composed of double-crossover (DX) molecules containing DNA hairpins. The use of 2D DNA crystals as a scaffolding potentially offers fundamental advantages over other self-assembly approaches for the precision, rigidity, and programmability of the assembled nanostructures, and the chemical selectivity of Nanogold labeling lets researchers use the full power of this to organize gold particles. The nanoparticles form precisely integrated components, which are covalently bonded to the DNA scaffolding. Scanning transmission electron microscopy (STEM) showed that the gold particles formed 2-D arrays with interparticle spacings of 4 and 64 nm.

DNA-Nanogold conjugates were prepared from trityl-protected 5'-thiol-modified C6 oligonucleotides, which were deprotected and reacted with Monomaleimido Nanogold: 6 nmol of deprotected oligonucleotide were combined with 6-12 nmol of Monomaleimido Nanogold in 200 microliters of reaction buffer (20mM NaH2PO4, 150mM NaCl, 1mMEDTA, pH 6.8): after incubation at room temperature for 1 h, unreacted Nanogold was separated from the unreacted DNA and DNA-gold conjugate by addition of 3M sodium acetate (20 microliters) and ice cold absolute ethanol (500 microliters), incubation at -20°C for 1.2 h, then centrifugation at 4°C in a microcentrifuge (13,000 rpm) for 15 min. The yellow supernatant containing the free Au nanoparticles was removed, and the dark precipitate was dissolved in 90 microliters of water and injected into a C4 reversed-phase HPLC column. Chromatograms were developed at 0.8 ml/min with an acetonitrile gradient in 0.1M aqueous triethylammonium acetate at pH 6.5. DNA : Nanogold labeling stoichiometry of the purified conjugate was estimated spectroscopically to be very close to the desired 1:1 product. Reference:

Xiao, S.; Liu, F.; Rosen, A. E.; Hainfeld, J. F.; Seeman, N. C.; Musier-Forsyth, K., and Kiehl, R. A.: Selfassembly of metallic nanoparticle arrays by DNA scaffolding. J. Nanoparticle Res., 4, 313-17 (2002).

Abstract (courtesy of the Journal of Nanoparticle Research):
http://ipsapp009.lwwonline.com/content/getfile/5035/15/11/abstract.htm

A number of other publications have described the conjugation of Nanogold to oligonucleotide, using both Monomaleimido Nanogold to label via thiol groups, and Mono-Sulfo-NHS-Nanogold to label via aliphatic amines; a complete list of references is given in our web site, as well as suggested methods and protocols for labeling and isolating conjugates.

More information:

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Gold Enhancement can Improve your Results - and Enable New Ones

GoldEnhance works like silver enhancement, except that it deposits gold instead of silver. The use of gold instead of silver has advantages for several applications:
  • Gold Enhanced particles are more electron-dense than silver enhanced ones, for greater contrast in the transmission electron microscope, and superior back-scattered electron (BSE) detection in the scanning electron microscope. The process can be used to obtain highly uniform particles; for example, in the references below, gold enhancement was used with secondary Nanogold®-Fab' conjugates for pre-embedding immunoelectron microscopy:

    Ward, T. H.; Polishchuk, R. S.; Caplan, S.; Hirschberg, K., and Lippincott-Schwartz, J.: Maintenance of Golgi structure and function depends on the integrity of ER export. J. Cell Biol., 155, 557-70 (2001).

    Abstract (courtesy of the Journal of Cell Biology):
    http://www.jcb.org/cgi/content/abstract/155/4/557 Reprint (courtesy of the Journal of Cell Biology):
    http://www.jcb.org/cgi/reprint/155/4/557.pdf

    Ethell, I. M.; Hagihara, K.; Miura, Y.; Irie, F., and Yamaguchi, Y.; Synbindin, A novel syndecan-2-binding protein in neuronal dendritic spines. J. Cell Biol., 151, 53-68 (2000).

    Abstract (courtesy of the Journal of Cell Biology):
    http://www.jcb.org/cgi/content/abstract/151/1/53 Reprint (courtesy of the Journal of Cell Biology):
    http://www.jcb.org/cgi/reprint/151/1/53.pdf

  • Gold enhancement is impervious to osmium etching; if you are having this problem, gold enhancement provides an alternative enhancement method, without the requirement for additional gold toning steps.

  • GoldEnhance is more selective than silver enhancement in the presence of components that react with silver ions, or promote non-specific silver deposition. For example, silver-enhanced immunogold labeling of specimens on metal substrates is problematic: smaller gold particles produce much more successful labeling, but the metal substrates promote severe etching of silver enhancement by osmium. Owen and co-workers have found that GoldEnhance is not affected, and successfully labeled vinculin in the focal adhesion sites of fibroblasts using 5 nm gold with gold enhancement. 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).

    Abstract (Courtesy of Ideal):
    http://www.idealibrary.com/links/doi/10.1006/cbir.2001.0846 Other publications by the authors:
    http://www.aofoundation.org/ari/research/interface/publications.shtml

    GoldEnhance may also be used in the presence of chloride buffers without precipitation problems.

  • GoldEnhance produces very clear signals with low background for light microscopy, and has proven particularly valuable in in situ hybridization detection. For example, the brightfield chromogenic Her-2/neu gene amplification assay developed by Tubbs and co-workers uses Nanogold-streptavidin with gold enhancement to generate a dense black signal, which is read in the light microscope. Unlike fluorescence in situ hybridization (FISH), this method allows simultaneous visualization of the underlying morphology, and the black stain is permanent, does not photobleach, and is easily distinguished from other commonly used stains. Reference:

    Tubbs, R.; Pettay, J.; Skacel, M.; Powell, R.; Stoler, M.; Roche, P., and Hainfeld, J.: Gold-Facilitated in Situ Hybridization: A Bright-Field Autometallographic Alternative to Fluorescence in Situ Hybridization for Detection of HER-2/neu Gene Amplification. Am. J. Pathol., 160, 1589-1595 (2002).

    Abstract (courtesy of the American Journal of Pathology):
    http://ajp.amjpathol.org/cgi/content/abstract/160/5/1589

More information about gold enhancement:

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Nanogold Labeling for Preembedding, Postembedding and Cryosections

Nanogold® -Fab' fragments are only about one-third the size of whole IgG molecules; the small size of Nanogold conjugates combined with their minimal background staining makes them suitable for a wide range of immunoelectron microscopy labeling techniques. The following references describe some recent protocols.

Hebert, Grondin and co-workers used pre-embedding immunoelectron microscopy with Nanogold conjugates and HQ Silver to study subcellular distribution of dual leucine zipper-bearing kinase b(DLK) at the ultrastructural level by immunoEM in NIH3T3 and COS-1 cells undergoing apoptosis. They differentiated the localization of higher MW oligomers from lower MW derivatives, thus helping to elucidate the mechanism underlying DLK oligomerization. Cells were rinsed twice with phosphate-buffered saline (PBS), fixed in paraformaldehyde-lysine-periodate fixative for 4 h at room temperature, then permeabilized with saponin and incubated, in sequence, with DLK antiserum followed by Nanogold-labeled goat anti-rabbit antibodies. After further fixation in 1.3% glutaraldehyde, silver enhancement (HQ Silver), and postfixation in 1% osmium tetroxide, 1% potassium ferrocyanide, the cells were embedded in situ with Poly/Bed® 812 epoxy resin (Polysciences, Inc., Warrington, PA). Sections were cut to 70 nm, and poststained with uranyl acetate and lead citrate. Reference:

Hebert, S. S.; Daviau, A.; Grondin, G.; Latreille, M.; Aubin, R. A.; and Blouin, R.: The mixed lineage kinase DLK is oligomerized by tissue transglutaminase during apoptosis. J. Biol. Chem., 275, 32482-90 (2000).

Abstract (courtesy of the Journal of Biological Chemistry):
http://www.jbc.org/cgi/content/abstract/275/42/32482 Reprint (courtesy of the Journal of Biological Chemistry):
http://www.jbc.org/cgi/reprint/275/42/32482.pdf

Bean and co-workers describe an effective postembedding method to localize the ATPase Hrs-2, helping to show that it regulates endocytosis and suggesting that it provides communication between endo- and exocytic processes. Rats were perfused with 3% paraformaldehyde, 0.1% glutaraldehyde, 0.2% picric acid in 0.1 M phosphate buffer. Brains were post-fixed in situ and sectioned on a vibratome. Tissues were embedded in Lowicryl and thin sectioned. Embedded tissues were incubated with Hrs-2 antibody (10) (1:200), Nanogold-Fab' anti-rabbit secondary antibody, and silver intensified. Cells were examined on an electron microscope (Jeol) and photographed. Reference:

Bean, A. J.; Davanger, S.; Chou, M. F.; Gerhardt, B.; Tsujimoto, S.; and Chang, Y.: Hrs-2 regulates receptor-mediated endocytosis via interactions with Eps15. J. Biol. Chem., 275, 15271-8 (2000).

Abstract (courtesy of the Journal of Biological Chemistry):
http://www.jbc.org/cgi/content/abstract/275/20/15271 Reprint (courtesy of the Journal of Biological Chemistry):
http://www.jbc.org/cgi/reprint/275/20/15271.pdf

Legare and group compare two different approaches for labeling cryosections prior to embedding, one using protein A-6nm gold, one using Nanogold, and find dense labeling with Nanogold. They use these methods investigate the mechanism by which the Leishmania ATP-binding cassette (ABC) transporter protein, PGPA, confers resistance to antimony and other metals (antimony is the first line drug against Leishmania). For immunoelectron microscopy, cells were fixed with 4% or 2% formaldehyde, 0.05% glutaraldehyde in 0.1 M HEPES, pH 7.0, for 60 min on ice. Fixed Leishmania cells were processed for ultrathin cryosection immunogold labeling; thawed frozen sections were reacted with rabbit anti-GFP antibodies or monoclonal mouse anti-HA antibody for 60 min each. Reactions were revealed using protein A-gold (6 nm) or goat anti-rabbit-IgG-Nanogold followed by silver enhancement, then stained with uranyl acetate and embedded in 1.8 % methylcellulose.

Legare, D.; Richard, D.; Mukhopadhyay, R.; Stierhof, Y.-D.; Rosen, B. P.; Haimeur, A.; Papadopoulou, B., and Ouellette, M.: The Leishmania ATP-binding cassette protein PGPA is an intracellular metal-thiol transporter ATPase. J. Biol. Chem., 276, 26301-7 (2001).

Abstract (courtesy of the Journal of Biological Chemistry):
http://www.jbc.org/cgi/content/abstract/276/28/26301 Reprint (courtesy of the Journal of Biological Chemistry):
http://www.jbc.org/cgi/reprint/276/28/26301.pdf

More information on Nanogold labeling and silver enhancement:

You can find a comprehensive collection of established protocols in the recently published book, "Gold and Silver Staining: Techniques in Molecular Morphology" edited by Gerhard W. Hacker and Jiang Gu (CRC Press, Boca Raton, FL).

More information (CRC Press):
http://www.crcpress.com/shopping_cart/products/product_detail.asp?sku=1392&parent_id=&pc=/shopping_cart/search/search.asp!

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How Do I Know if my Nanogold® Labeling Worked?

We are frequently asked what are the extinction coefficients of Nanogold®, and also for UV/visible spectra of our Nanogold and undecagold labeling reagents and their conjugates. All this information, as well as suggestions for optimizing your labeling reaction and for separating gold conjugates from other species, are given in our online Guide to Gold Cluster Labeling. We list extinction coefficients at 280 nm (300,000 M-1cm-1 for Nanogold) and 420 nm (110,000 for Nanogold) because these are most useful for calculating labeling of proteins, and the Guide includes detailed instructions for using these to calculate labeling. In addition, the spectra are shown from 260 nm to 820 nm; you can use them to estimate extinction coefficients at other wavelengths.

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Other Recent Publications

Grassme and co-workers used Nanogold® in scanning electron microscopy (SEM) to show that acid sphingomyelinase (ASM)-released ceramide is essential for clustering of CD95 and apoptosis induction. Cells were immobilized on plastic coverslips, stimulated, fixed in 4% PFA/PBS, blocked with a 15-min incubation with 0.1% BSA-C/PBS, and incubated for 60 min each with mouse anti-ASM and a Nanogold-coupled goat anti-mouse antibody (Nanoprobes, NY). Samples were postfixed in 4% PFA/PBS, dehydrated, critical-point-dried from CO2, sputter-coated with 1 nm Cr and examined at 10 kV accelerating voltage.

Reference:

Grassme, H.; Jekle, A.; Riehle, A.; Schwarz, H.; Berger, J.; Sandhoff, K.; Kolesnick, R, and Gulbins, E.: CD95 signaling via ceramide-rich membrane rafts. J. Biol. Chem., 276, 20589-96 (2001).

Abstract (courtesy of the Journal of Biological Chemistry):
http://www.jbc.org/cgi/content/abstract/276/23/20589 Reprint (courtesy of the Journal of Biological Chemistry):
http://www.jbc.org/cgi/reprint/276/23/20589.pdf

Meanwhile, Le and group use postembedding double immunogold labeling with Nanogold and 12 nm or 18 nm colloidal gold labeling to differentiate the localizations of Autocrine motility factor receptor (AMF-R) and caveolin. The authors used pulsed (electroporetic) biotinylated AMF (bAMF) followed by Nanogold-streptavidin and silver enhancement to localize AMF receptors, then a second labeling with polyclonal rabbit anti-caveolin followed by 12 nm gold-labeled anti-rabbit secondary to localize caveolin.

References:

Le, P. U.; Guay, G.; Altschuler, Y., and Nabi, I. R.: Caveolin-1 is a negative regulator of caveolae-mediated endocytosis to the endoplasmic reticulum. J. Biol. Chem., 277, 3371-9 (2002).

Abstract (courtesy of the Journal of Biological Chemistry):
http://www.jbc.org/cgi/content/abstract/277/5/3371

Le, P. U.; Benlimame, N.; Lagana, A.; Raz, A., and Nabi, I. R.: Clathrin-mediated endocytosis and recycling of autocrine motility factor receptor to fibronectin fibrils is a limiting factor for NIH-3T3 cell motility. J. Cell Sci., 113, 3227-40 (2000).

Abstract (courtesy of the Journal of Cell Science):
http://jcs.biologists.org/cgi/content/abstract/113/18/3227 Reprint (courtesy of the Journal of Cell Science):
http://jcs.biologists.org/cgi/reprint/113/18/3227.pdf

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