Updated: June 12, 2002
N A N O P R O B E S E - N E W S
Vol. 3, No. 6 June 12, 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 tech@nanoprobes.com.
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Tolstonog and co-workers used a double-labeling procedure with Nanogold®-labeled antibodies to demonstrate the stable association of cytoplasmic intermediate filaments (cIFs) with nuclear lamins, indicating that they are complementary or even integral components of the karyoskeleton.
Two Nanogold-labeled antibodies were used for labeling. First, nuclear lamins were labeled with a goat anti-lamin primary antibody followed by Nanogold-conjugated IgG rabbit anti-goat secondary antibody; the gold particle were then silver enhanced (using R-Gent-SE-ME solution, an Aurion silver enhancer which works equally well with Nanogold). After this, in a second labeling procedure, a goat anti-vimentin antibody, labeled using Monomaleimido Nanogold, was used to label intermediate filaments. When both labeling procedures were complete, a second silver enhancement step was carried out for 45 minutes (R-Gent-SE-ME). TEM examination of the labeled cells showed that the nuclear lamins were labeled with approximately 50 nm silver-enhanced gold, while the intermediate filaments were labeled with smaller silver-enhanced gold particles. The minimum separation between the two labeling sites was found to be about 30 nm.
Reference:
Tolstonog, G. V.; Sabasch, M., and Traub, P.: Cytoplasmic Intermediate Filaments Are Stably Associated with Nuclear Matrices and Potentially Modulate Their DNA-Binding Function. DNA Cell Biol., 21, 213-39 (2002).
Abstract (Medline):
http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=12015898&dopt=Abstract
A previous issue of this newsletter (March 1, 2001) focused on double labeling. Articles are included on double labeling with different sized golds, combining gold labeling and silver enhancement with enzymatic labeling, and differentiating targets using different degrees of silver enhancement:
Double labeling issue: www.nanoprobes.com/Vol2_Iss2.html
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In a recent paper, Stoffel and co-workers used cationic 15 nm colloidal gold to determine the distribution and density of surface sites on boar ejaculated and epididymal spermatozoa. Labeling was conducted at pH 2.5 (the low pH favors cationization of the gold).
Reference:
Stoffel, M. H.; Busato, A., and Friess, A. E.: Density and distribution of anionic sites on boar ejaculated and epididymal spermatozoa. Histochem. Cell Biol., 117, 441-5 (2002).
Abstract (Medline):
http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=12029491&dopt=Abstract
You may not be aware that Nanogold (or undecagold) can also be supplied in a positively ionizing form with multiple amino- groups, or a negatively ionizing form with carboxyl groups, that can assume positive or negative charges. You can use these reagents to label or stain features in your specimens that have the opposite charge. Prescianotto-Baschong and co-workers have used positively charged Nanogold to label elements of the yeast endocytic pathway:
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:
In addition, these can be the building blocks for novel nanostructured materials. In one such application, DNA strands were used as templates to deposit positively charged Nanogold particles; these may then be linked to form molecular wires by the autometallographic deposition of silver (silver enhancement) or gold (gold enhancement):
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Autometallography can detect more than gold nanoparticles. Gorm Danscher, one of the originators of the method, has it to localize deposits of several metals directly in cells and tissues. Zinc may be detected by treatment with sodium selenide to convert the zinc to zinc selenide, followed by silver acetate autometallography (Wang et al, 2002); bismuth may be detected autometallographically (Danscher et al, 2000); and similar methods exist for other heavy metals (Stoltenberg and Danscher, 2000). More recently, autometallography has been used to trace gold liberated in vivo from gold implants (Danscher, 2002).
References:
Wang, Z. Y.; Li, J.Y.; Danscher, G., and Dahlstrom, A.: Localization of zinc-enriched neurons in the mouse peripheral sympathetic system. Brain Res., 928, 165-74 (2002).
Abstract (Medline):
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=11844484&dopt=Abstract
Danscher, G.; Stoltenberg, M.; Kemp, K., and Pamphlett, R.: Bismuth autometallography: protocol, specificity, and differentiation. J. Histochem. Cytochem., 48, 1503 (2000).
Abstract (Journal of Histochemistry and Cytochemistry):
http://www.jhc.org/cgi/content/abstract/48/11/1503
Stoltenberg, M., and Danscher, G.: Histochemical differentiation of autometallographically traceable metals (Au, Ag, Hg, Bi, Zn): protocols for chemical removal of separate Autometallographic metal clusters in Epon sections. Histochem. J., 32, 645-52 (2000).
Abstract (Medline):
http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=11272803&dopt=Abstract
Danscher, G.: In vivo liberation of gold ions from gold implants. Autometallographic tracing of gold in cells adjacent to metallic gold. Histochem. Cell Biol., 117, 447-52 (2002).
Abstract (Medline):
http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=12029492&dopt=Abstract
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Our Guide to Gold Cluster Labeling includes sections that will tell you how to get the best results from every step of the gold labeling process, as well as how to find out how well your gold labeling reaction went and find out what proportion of your biomolecules were labeled.
There are a number of technical questions that our customers frequently ask. We plan to update the instructions and technical information for many of our products in the near future, but in the meantime, here are the answers to some of your most common questions:
Q: What is the molecular weight of Nanogold®?
A: Approximately 15,000
Q: How many functional groups do your labeling reagents contain? How do you know?
A: Close to one. Our labeling reagents are prepared from gold clusters with close to one amine, which are separated from non-functional and more heavily functionalized species by ion exchange chromatography.
Q: what is the concentration of Nanogold® conjugates?
A: All Nanogold conjugates (IgG, Fab' and streptavidin) are supplied at a concentration of 80 micrograms/mL.
Q: Do you have a gold reagent for labeling polyhistidine tags?
A: We are currently developing and testing this product. We expect that it will be available in 2-3 months. When it is introduced, it will be announced on our web site; if it's not on our web site, it is not available.
For further technical help, please visit our technical support pages:
- Technical Support:
- Product literature citations:
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Koberna and co-workers correlate fluorescence and immunoelectron microscopy to study ribosomal genes. They observed that on the light microscopic (LM) level, ribosomal genes in HeLa cells map into 10-40 foci; transcription activity is associated with most foci. Each nucleolar focus observed by LM corresponds, on the electron microscopic (EM) level, to an individual fibrillar center (FC) and surrounding dense fibrillar components (DFCs). In this case, 'simultaneous' visualization was accomplished by using a gold-conjugated secondary antibody followed by a fluorescently labeled tertiary.
Reference:
Koberna, K.; Malinsky, J.; Pliss, A.; Masata, M,; Vecerova, J.; Fialova, M.; Bednar, J., and Raska, I.: Ribosomal genes in focus: new transcripts label the dense fibrillar components and form clusters indicative of "Christmas trees" in situ. J. Cell Biol., 157, 743-8 (2002).
Abstract (Journal of Cell Biology):
http://www.jcb.org/cgi/content/abstract/157/5/743
Kaur and co-workers have developed an ELISA-based method for rapid selection of optimal blocking agents to be used in antigen quantification by immunogold labeling electron microscopy. Casein, skim milk, BSA from two sources, acetylated BSA, fish skin gelatin, horse serum, and goat serum were tested for their ability to block nonspecific binding of antibody to recombinant Vitreoscilla hemoglobin (VHb) antigen expressed in Escherichia coli cells by ELISA, dot blot and western blot methods; when the results were confirmed by quantitative immunogold labeling transmission electron microscopy (TEM), the results demonstrated that ELISA was most accurate in predicting the most efficient blocking agent for TEM.
Reference:
Kaur, R.; Dikshit, K. L.; and Raje, M.: Optimization of immunogold labeling TEM: an ELISA-based method for evaluation of blocking agents for quantitative detection of antigen. J. Histochem. Cytochem., 50, 863-73 (2002).
Abstract (Journal of Histochemistry and Cytochemistry):
http://www.jhc.org/cgi/content/abstract/50/6/863
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