Updated: April 2, 2001
N A N O P R O B E S E - N E W S
Vol. 2, No. 3 April 2, 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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Molecuar 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 about it in Nature Biotechnology:
Dubertret, B., Calame, M., and Libchaber, A.; Nat. Biotechnol., 19, 365-370 (2001).
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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Thought Nanogold®-Fab' was the smallest gold immunoprobe you could get? If you are using Fv fragments, you can make a smaller probe by labeling these with Mono-Sulfo-NHS-Nanogold. See how in this paper from Ribrioux and co-workers:
Ribrioux, S., Kleymann, G., Haase, W., Heitmann, K., Ostermeier, C., and Michel, H.; J. Histochem. Cytochem., 44, 207-213 (1996).
Abstract: http://www.jhc.org/cgi/content/abstract/44/3/207
Protocols for Mono-Sulfo-NHS-Nanogold: www.nanoprobes.com/Inf2025.html
References for Mono-Sulfo-NHS-Nanogold: www.nanoprobes.com/Refnglr.html#2025new
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If you are familiar with positively charged colloidal gold (such as polylysine-gold), you may not be aware that Nanogold® (or undecagold) can be supplied with multiple amino- groups or carboxyl groups so that it can assume positive or negative charge. You can use these reagents to label or stain features in your specimens that have the opposite charge. For example, positively charged Nanogold® can stain DNA. In these two papers, Prescianotto-Baschong and co-workers describe how they labeled elements of the yeast endocytic pathway using positively charged Nanogold®:
1.Seron, K., Tieaho, V., Prescianotto-Baschong, C., Aust, T., Blondel, M. O., Guillard, P., Devilliers, G., Rossanese, O. W., Glick, B. S., Riezman, H., Keranen, S., and Haguenauer-Tapis, R.: A yeast t-SNARE involved in endocytosis; Mol. Biol. Cell, 9, 2873 (1998).
Prescianotto-Baschong, C., and Riezman, H.: Morphology of the yeast endocytic pathway. Mol. Cell Biol., 9, 173-189 (1998).
More information:
Catalog information (Nanogold): www.nanoprobes.com/LabRgts.html#charged
Catalog information (Undecagold): www.nanoprobes.com/LabRgts.html#charged
Product information - positively charged Nanogold: www.nanoprobes.com/Inf2022.html
Product information - negatively charged Nanogold: www.nanoprobes.com/Inf2023.html
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Because Nanogold® is relatively small, you can make it difficult to visualize by applying additional heavy metal based stains. However, if you are negative staining - staining the regions between or around the edges of your specimens - you have the option of using a lighter stain. NanoVan is based on vanadium, and therefore 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. For an example of NanoVan in use, see Gregori, L., et al.; J. Biol. Chem., 272, 58-62 (1997).
Reprint (pdf): http://www.jbc.org/cgi/reprint/272/1/58.pdf
Our paper from M & M 94: www.nanoprobes.com/MSANV.html
Catalog Info and details: www.nanoprobes.com/Nstain.html
Other references: www.nanoprobes.com/Refstain.html
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Schéfer and co-workers used Nanogold® antibodies and streptavidin to label DNA, with gold enhancement, in high-resolution electron microscopy for the morphometric analysis of DNA in human spermatocytes:
Weipoltshammer, K.; Schéfer, C.; Almeder, M., and Wachtler, F.; Histochem. Cell. Biol., 114, 489-95 (2000).
Abstract: http://link.springer-ny.com/link/service/journals/00418/bibs/0114006/01140489.htm
Stewart and co-workers used Nanogold® to localize the two dimers of L7/L12 within the structure of the 70S ribosome, using two reconstitution approaches together with cryo-electron microscopy and single particle reconstruction. Protein L7/L12 was reduced with 1 % mercaptoethanol and labeled with Monomaleimido Nanogold®:
Abstract: http://www.jbc.org/cgi/content/abstract/M008430200v1
Accepted Manuscript: http://www.jbc.org/cgi/reprint/M008430200v1.pdf
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