Updated: October 9, 2001

N A N O P R O B E S     E - N E W S

Vol. 2, No. 9          October 9, 2001

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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Research Application: Aldehyde Gold for Molecular Labeling

Our abstract from the 1995 Annual Meeting of the Microscopy Society of America describes the preparation and use of gold clusters functionalized with a new reactive moiety: multiple aldehyde groups. Aldehydes, unlike Sulfo-NHS esters and other readily hydrolyzed amine-reactive functionalities, are stable in aqueous solution, and this approach may have benefits in some systems.

Our paper: www.nanoprobes.com/MSA95Ald.html

We have rearranged our "Applications" page into two sections to make searching easier: product applications, which describe new applications and uses for our current product line, and research applications, which describe the preparation and use of new or experimental reagents which we do not yet offer as products. Also this month, see our research application paper describing a covalently linked 10 nm gold-Fab' immunoprobe.

Applications page: www.nanoprobes.com/Applic.html
10 nm covalent gold probe: www.nanoprobes.com/MSA99LG.html

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Nanogold® Quenched Molecular Beacons

Molecular beacons are hairpin loops of DNA, with a fluorescent group at one end and a quencher at the other: when they bind to their target, they open, the fluorophore and the quencher move apart, and fluorescent signal appears. The conventional quencher is DABCYL; however, Nanogold® has been found to be much more effective, and can improve the "signal-to-noise ratio" (the ratio of fluorescence intensity when the beacon is open to when it is closed) from 100 to up to several thousand. Read their Nature Biotechnology paper:

Dubertret, B., Calame, M., and Libchaber, A. Nat. Biotechnol., 2001, 19, 365-370.
Abstract (requires registration): http://www.nature.com/nbtlink/v19/n4/abs/nbt0401_365.html

More information about the conjugation of Nanogold to oligonucleotides:

Technical help with DNA labeling: www.nanoprobes.com/TechNGlr.html#DNA
Application note: RNA labeling: www.nanoprobes.com/App2.html
References: www.nanoprobes.com/RefTopNG.html#Ndna
Catalog information on Nanogold labeling reagents: www.nanoprobes.com/LabRgts.html

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Make your own Double Labeled Probes

When you attach Nanogold® to your probes, you are not restricted to *only* gold labeling. You can attach a second label to the probe, and then use the double-labeled probe to label your antigens for observation by two different, complementary imaging or detection modalities. Couple an enzyme as well as Nanogold and detect your antigens with chemiluminescent or chromogenic substrates. Use an amine-reactive fluorescent labeling reagent to put a novel fluorophore onto an antibody or protein that you have labeled with Monomaleimido-Nanogold. Once you have attached the gold, the link is stable, and you can couple a second label under a wide variety of reaction conditions.

Information and pricing: www.nanoprobes.com/LabRgts.html
References: www.nanoprobes.com/Refnglr.html
Technical Help: www.nanoprobes.com/TechNGlr.html

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Double labeling with Different Sized Golds

You can also double label with different sized gold particles, with or without silver enhancement. Takizawa and Robinson have demonstrated that the dense labeling of granules with Nanogold positive for DAF were easily distinguished from 10 nm colloidal gold labeled lactoferrin-containing granules; reference:

Takizawa, T. and Robinson, J.M.: J. Histochem. Cytochem., 42, 1615-1623 (1994).
Abstract: http://www.jhc.org/cgi/content/abstract/42/12/1615

Matsubara and co-workers stained GluR2/3 with dense fine deposits of silver enhanced Nanogold staining that were easily distinguishable from 30 nm colloidal gold immunostaining of glutamate. Nusser and colleagues used postembedding (freeze-substituted, Lowicryl embedded ultrathin sections) with silver amplified for 15 nm gold (giving large 40 nm particles), targeted to GABA, and silver intensified Nanogold, targeted to GABAA receptors, on the same section to simultaneously view the distributions of these targets in cerebellar granule cells. References:

Matsubara, A., J.H. Laake, S. Davanger, S.-I. Usami, and O.P. Otterson. 1996. J. Neuroscience, 16, 4457-4467.
Abstract: http://www.jneurosci.org/cgi/content/abstract/16/14/4457

Nusser, Z., J.D.B. Roberts, A. Baude, J.G. Richards, and P. Somogyi: J. Neuroscience, 15, 2948-2960 (1995).
Abstract: http://www.jneurosci.org/cgi/content/abstract/15/4/2948

References for other applications of Nanogold: www.nanoprobes.com/RefTopNG.html
Technical Help for Nanogold conjugates: www.nanoprobes.com/TechNGAb.html

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Nanoprobes Products Now Available from Molecular Probes

Nanoprobes and Molecular Probes, Incorporated have signed a distribution agreement which lets Molecular Probes include Nanogold® labeling reagents, a select range of Nanogold-antibody conjugates, and silver enhancement reagents as part of their catalog. Molecular Probes is a leader in the development of fluorescent probes, and the companies anticipate that by including these products in the widely distributed Molecular Probes Handbook of Fluorescent Probes, these probes will be introduced to more customers, and will enable microscopists who routinely use Molecular Probes' fluorescent labeling reagents to conveniently order high-quality electron microscopy labeling reagents from the same source.

Press release: www.nanoprobes.com/News01.html#MolProbesAgr
Molecular Probes: http://www.probes.com
Handbook of Fluorescent Probes: http://www.probes.com/handbook/

We are also pleased to announce that we have received a new Small Business Innovation Research grant from the National Institute of General Medical Sciences (NIGMS), part of NIH. The grant of $ 106,000 for six months, effective September 24, 2001, will be used to research and develop a novel enzymatically mediated metal deposition reaction for biological detection and staining. The new detection system utilizes the selective deposition of metal from solution onto specific sites within cells and tissues labeled with an enzyme-linked antibody or other biomolecule conjugate targeted to the sites of interest. It has been found to give high sensitivity combined with high spatial and detail resolution within samples, making it useful both for the identification and quantitation of cellular features for disease identification, and for microscopic and ultrastructural investigation. This technology will be developed for use as a general staining and detection method for the immunohistochemical staining of proteins, and also in situ hybridization detection of genetic changes in cells and tissue sections.

Press release: www.nanoprobes.com/News01.html#1R43GM64257
Nanoprobes news updates for 2001: www.nanoprobes.com/News01.html

We have also recently added two new staff, Robert A. Hausner (Controller) and Victoria A. Kowalsky (Office Assistant) to help with order processing and financial administration.

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Recent Publications

Sohda, Ikehara and co-workers used Nanogold®-labeled secondary antibodies with silver enhancement to determine the distribution of a novel Golgi protein, GCP60, that interacts with the membrane protein giantin. Using immunofluorescence with ALEXA-labeled secondary antibodies in addition to immunoEM, this group found that the new 528 amino acid, 60 kDa protein, was co-localized with giantin in the Golgi complex. The paper is currently in press in the Journal of Biological Chemistry:

Abstract: http://www.jbc.org/cgi/content/abstract/M108961200v1
Accepted manuscript: http://www.jbc.org/cgi/reprint/M108961200v1.pdf

John M. Robinson and Dale Vandr review antigen retrieval, and in particular its enhancement with sodium dodecylsulfate, in their recent paper in Histochemistry and Cell Biology. The extent to which the antigen is accessible and recognizable for labeling is another important factor in successful immunolabeling. The article refers principally to fluorescence microscopy,but the discussion also applies equally to FluoroNanogold probes.

Abstract: http://link.springer-ny.com/link/service/journals/00418/bibs/1116002/11160119.htm

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