Updated: November 7, 2001
N A N O P R O B E S E - N E W S
Vol. 2, No. 10 November 7, 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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Because Nanogold® and undecagold are chemically cross-linked, like fluorescent labels, rather than adsorbed like colloidal gold, you can conjugate them to a wide variety of other molecules in addition to antibodies and proteins. Many other molecules that cannot be labeled with conventional colloidal gold can readily be labeled with Nanogold. Large or small, water-soluble or best handled in other solvents, all that is needed for successful gold-labeling is an accessible thiol (sulfhydryl) or amino- group. And with Monoamino-Nanogold, you can use many other heterobifunctional cross-linking reagents to link Nanogold to other functional groups.
Some examples:
Undecagold can even be used as an isomorphous heavy atom replacement to derivatize proteins for X-ray crystallographic studies:
References: www.nanoprobes.com/RefTopUG.html#Uiso
How do you get the best results from labeling, and how do you know whether it worked? Our Guide to Gold Cluster Labeling shows you the process step-by-step, with all the factors that can affect your labeling reaction described and explained. The Guide also includes the UV/visible spectra of Nanogold and undecagold both unconjugated and conjugated, and shows you how to calculate your labeling exactly.
Guide to Gold Cluster Labeling: www.nanoprobes.com/LGuide.html
Spectra and labeling calculations: www.nanoprobes.com/LGuide4.html
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A recent discussion on the MSA microscopy listserver generated many ideas and suggestions for optimizing your TEM immmunolabeling, as well as explanations for why some antibodies work better than others. Read about the factors that can affect labeling performance of both primary and secondary antibodies, and how to test your labeling effectively before proceeding to a full TEM experiment.
Full details: www.nanoprobes.com/TechIEM.html
More technical help for Nanoprobes products: www.nanoprobes.com/Tech.html
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FluoroNanogold is a unique probe containing both Nanogold® and fluorescein. You can use it for truly correlative labeling on the cellular and macromolecular level, or for checking your labeling by fluorescence before EM processing.
Catalog information: www.nanoprobes.com/FluoroN.html
Because FluoroNanogold combines two labels which are frequently used in different concentrations under different conditions, some optimization may be required to obtain the best performance in your experiments. For tips and tricks on FluoroNanogold labeling, and how to obtain the cleanest signal, visit our technical help page:
Technical help for FluoroNanogold: www.nanoprobes.com/TechFN.html
Original papers:
- Powell, R. D.; Halsey, C. M. R.; Spector, D. L.; Kaurin, S. L.; McCann, J.;, and Hainfeld, J. F. A covalent fluorescent-gold immunoprobe: "simultaneous" detection of a pre-mRNA splicing factor by light and electron microscopy. J. Histochem. Cytochem., 45, 947-956 (1997).
Reprint: http://www.jhc.org/cgi/reprint/45/7/947.pdf
- Robinson, J. M., and Vandr, D. D. Efficient immunocytochemical labeling of leukocyte microtubules with FluoroNanogold: An important tool for correlative microscopy. J. Histochem. Cytochem., 45, 631-642 (1997).
Reprint: http://www.jhc.org/cgi/reprint/45/5/631.pdf
Takizawa, T.; Suzuki, K., and Robinson, J. M.: Correlative Microscopy Using FluoroNanogold on Ultrathin Cryosections: Proof of Principle; J. Histochem. Cytochem., 46, 1097-1102 (1998).
Reprint: http://www.jhc.org/cgi/reprint/46/10/1097.pdf
On our web site, you can also see our paper from the Microscopy Society of America Annual Meeting, 1999, describing the use of combined Cy3 and Nanogold probes for immunolabeling and in situ hybridization. Combined fluorescent and gold probes with other fluorophores are now under development for future commercial introduction.
Paper: www.nanoprobes.com/MSAFN99.html
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A complete list of literature citations for our products is available on our web site. You can view references sorted in two ways: by product, or by label and application. The entire section is now linked from a single index page:
www.nanoprobes.com/Ref.html
We are committed to linking our reference pages to as much content as is available. For example, all references in Proceedings of the National Academy of Sciences of the USA since 1996 are now linked directly to both the online abstract and the full reprint of the paper in PDF format. Citations are linked to abstracts when these are available.
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The seven-subunit Arp2/3 complex choreographs the formation of branched actin networks at the leading edge of migrating cells. When activated by Wiskott-Aldrich Syndrome protein (WASp), the Arp2/3 complex initiates actin filament branches from the sides of existing filaments. Hanein and co-workers used Nanogold® to label Scar-WA-Cys, a WASp analog modified with an N-terminal cysteine, and then used the labeled peptide to localize the WASp binding site in cryoelectron microscopy reconstructions of the activated Arp2/3 complex.
Reference:
Volkmann, N.; Amann, K. J.; Stoilova-McPhie, S.; Egile, C.; Winter, D. C.; Hazelwood, L.; Heuser, J. E.; Li, R.; Pollard, T. D., and Hanein, D.: Structure of Arp2/3 Complex in Its Activated State and in Actin Filament Branch Junctions. Science, 293, 2456-9 (2001).
Abstract: http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=11533442&dopt=Abstract
In situ PCR is reviewed by Gerard Nuovo in the current issue of the Journal of Histochemistry and Cytochemistry. This review describes a methodology that allows co-in situ localization of two nucleic acid targets or a DNA/RNA sequence and a protein in paraffin-embedded, formalin-fixed tissue.
Reference:
Nuovo, G. J.: Co-labeling Using In Situ PCR: A Review. J. Histochem. Cytochem., 49, 1329-1340 (2001).
Abstract: http://www.jhc.org/cgi/content/abstract/49/11/1329
Capani and co-workers describe the use of eosin-labeled phalloidin to localize actin filaments; labeling can be directly visualized by fluorescence microscopy, then photooxidation of DAB is used to convert the staining to a signal for electron microscopy.
Reference:
Capani, F.; Deerinck, T. J.; Ellisman, M. H.; Bushong, E.; Bobik, M., and Martone, M. E.: PhalloidinEosin Followed by Photo-oxidation: A Novel Method for Localizing F-Actin at the Light and Electron Microscopic Levels. J. Histochem. Cytochem., 49, 1351-1362 (2001).
Abstract:
http://www.jhc.org/cgi/content/abstract/49/11/1351
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