Updated: March 11, 2003
N A N O P R O B E S E - N E W S
Vol. 4, No. 3 March 11, 2003
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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The wide variety of optical and electronic properties of gold clusters and nanoparticles give them many potential applications in nanotechnology, as components of novel materials and molecular or macromolecular devices. Gold clusters with chemically selective reactivity possess another important advantage: they may be conjugated to specific sites within biological molecules where these properties impart useful functionality. The Nanogold® cluster, used in this manner, has produced several novel results. For example, Hamad-Schifferli and co-workers used amino-modified molecular beacons labeled with Mono-Sulfo-NHS Nanogold to achieve remote control of DNA hybridization, by means of the highly localized temperature rise produced by the conjugated Nanogold® particle inductively coupled to a pulsed radio frequency magnetic field. The effect was fully reversible and highly localized.
Reference:
Hamad-Schifferli, K.; Schwartz, J. J.; Santos, A. T.; Zhang, S., and Jacobson, J. M.: Remote electronic control of DNA hybridization through inductive coupling to an attached metal nanocrystal antenna. Nature, 415, 152-155 (2002).
Abstract (Medline):
LINK
Gold particles have been shown to be highly effective quenchers for molecular beacons. Dubertret et al have found Nanogold to be much more effective than the conventional DABCYL quencher, 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. Dulkeith and co-workers, investigating the effect on radiative and non-radiative fluorescence lifetimes for systems in which fluorophores were linked to metal nanoparticles, found both an increase in the radiative lifetime, and a decrease in the non-radiative lifetime, both of which imply that fluorescence quenching by attached gold particles is greater than predicted by Frster theory alone: even with 1 nm gold particles, a gold-fluorophore separation of 1 nm, gives about 99.8 % quenching.
References:
Dubertret, B., Calame, M., and Libchaber, A.: Single-mismatch detection using gold-quenched fluorescent oligonucleotides. Nat. Biotechnol., 19, 365-370 (2001).
Abstract (Medline):
LINK
Dulkeith, E.; Morteani, A. C.; Niedereichholz, T.; Klar, T. A.; Feldmann, J.; Levi, S. A.; van Veggel, F. C. J. M.; Reinhoudt, D. N.; Enschede, A. E.; Mller, M., and Gittins, D. I.: Fluorescence Quenching of Dye Molecules near Gold Nanoparticles: Radiative and Nonradiative Effects. Phys. Rev. Lett., 89, 203002 (2002).
Abstract (courtesy of Physical Review Letters):
LINK
Kiehl and co-workers report 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: DNA-Nanogold conjugates were prepared from trityl-protected 5'-thiol-modified C6 oligonucleotides, which were deprotected and reacted with Monomaleimido Nanogold. 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):
LINK
Nanoprobes is now working to bring the same chemically selective reactivity to larger gold particles. For some preliminary results, see our extended abstract from Microscopy & Microanalysis 99:
Our 1999 Microscopy & Microanalysis paper: www.nanoprobes.com/MSALG99.html
The organizing properties of biological molecules may also be used to assemble attached gold clusters into supramolecular arrays with a variety of configurations. For example, Nanogold®-labeled lipids may be used for form gold cluster-decorated liposomes with a variety of morphologies; called metallosomes, these novel structures may be starting points for novel nanostructured materials.
References:
Hainfeld, J. F.; Furuya, F. R., and Powell, R. D.: Metallosomes. J. Struct. Biol., 127, 152-160 (1999).
Abstract (courtesy of Science Direct):
LINK
Our 1996 Microscopy & Microanalysis paper: www.nanoprobes.com/MSA96lip.html
DNA strands decorated with positively charged Nanogold may form the basis for autometallographically generated molecular wires. Since current lithographic chip production techniques produce wires about 0.3 microns in diameter, molecular wires could offer large increases in packing density; for more details, see our extended abstract from Microscopy & Microanalysis 01.
Our 2001 Microscopy & Microanalysis paper: www.nanoprobes.com/MSADNA01.html
More information:
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The cross-linking rationale used for Nanogold® labeling also means that it can be conjugated to smaller fragments of antibodies, giving both improved penetration into specimens, higher density labeling, and higher resolution localization.
Uchiyama and co-workers used Nanogold labeling of Golgi marker proteins to help characterize and elucidate the role of a novel essential factor for p97/p47-mediated membrane fusion, named VCIP135 (valosin-containing protein [VCP][p97]/p47 complex-interacting protein, p135), in Golgi reassembly. Immunofluorescence studies of the effects of anti-VCIP135 antibodies on Golgi organization in living cells, combined with binding experiments with related components, demonstrated that VCIP135 functions in Golgi and ER assembly. Immunoelectron microscopy was then used to investigate changes in the ultrastructure of Golgi in anti-VCIP135 antibody-injected cells. Highly organized Golgi complexes were lost: stacked cisternae were rarely observed, and the number of tubular/fenestrated structures was greatly increased. amount of vesicles was not changed. Similar morphological changes were observed after the injection of anti-p47 antibodies. Since the injection of the anti-VCIP135 antibodies at the same concentration into interphase cells caused no obvious changes in Golgi organization, these morphological changes were most likely caused by the inhibition of reassembly at the end of mitosis. These data show that VCIP135 functions in the reassembly of highly organized Golgi structures in living cells as does p47, indicating that p97-mediated fusion is required for Golgi reassembly.
Cells were fixed with PLP fixative (2% formaldehyde, 0.01 M periodate, 0.075 M lysineHCl in 0.075 M phosphate buffer, pH 7.4) for 2 h at RT, permeabilized with 0.01% saponin and immunolabeled using anti-GalT or anti-GM130 monoclonal primary antibodies and Nanogoldconjugated Fab secondary antibody fragments against mouse IgM + IgG. Nanogold was silver enhanced using HQ Silver for 0.5 to 4 min, and gold toned with 0.05% gold chloride.
Reference:
Uchiyama, K.; Jokitalo,E.; Kano, F.;Murata, M.; Zhang, X.; Canas, B.; Newman, R.; Rabouille, C.; Pappin, D.; Freemont, P., and Kondo, H.: VCIP135, a novel essential factor for p97/p47-mediated membrane fusion, is required for Golgi and ER assembly in vivo. J. Cell Biol., 159, 855-866 (2002).
Abstract (courtesy of the Journal of Cell Biology):
http://www.jcb.org/cgi/content/abstract/159/5/855
More information:
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We maintain a detailed technical help section on our web site with answers to frequently asked questions about how to use our products; we also explain the underlying technology of gold cluster labeling and differences compared with conventional colloidal gold in our online Guide to Gold Cluster Labeling.
If your question is not answered by these resources, and you need to contact us, then use the following guidelines to help us find your answer more quickly:
- If you are inquiring about gold labeling, remember that we use chemical cross-linking. In order to determine the feasibility of gold-labeling a specific molecule, we need to know the available chemical groups to which the gold label might be conjugated. We may not be familiar with the particular molecule you are working with, so sending us an illustration of the structure (or even a line of text showing the chemical connectivity) saves us the time spent looking it up.
- Our labeling reactions are similar to fluorescent or enzymatic labeling, and proceed through similar reactions and cross-linkers. Therefore, if you know of a prior reference where another group has conjugated your molecule with a fluorescent or enzymatic label, please include it - the reference will be very helpful because it helps suggest conditions and procedures which might be useful for gold labeling.
- The more information we receive, the more specific we can be with our answers. If you tell us what procedures you have used for labeling, isolating your conjugate, or preparing your specimens for microscopy, it helps us identify specific factors that might be affecting your result, and also helps to eliminate others from consideration. If you can provide information such as the molecular weight and optical density - or, even better, extinction coefficient - for the molecule you wish to label, we can suggest the best methods and media for separation.
Generally, we are currently restricting custom syntheses to well-established procedures that do not require novel chemistry. Commitments to other projects mean that the time and resources available for more speculative custom work are tightly restricted. However, we are always interested in hearing about new applications for our technology, and may be able to suggest other ways to proceed even if we can't fulfill a specific request.
More information:
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Our collaborators from the Cleveland Clinic Foundation will be presenting a poster at the upcoming US-Canadian Academy of pathology (USCAP) meeting in Washington DC, describing the latest developments in gold-facilitated in situ hybridization (GOLDFISH), in which Nanogold-streptavidin and GoldEnhance are used for the brighfield light microscope detection of Her-2/neu gene amplification detection. Look for the poster in the session on Breast Pathology on the afternoon of Tuesday, March 25.
More information:
Our expansion continues with the addition of Linda Schettini, our new Customer Service Representative, who joined us on March 10. She will be taking orders and answering your questions in the afternoons. With her help, we will be able to provide quicker and better service to our customers.
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Luby-Phelps and co-workers describe a procedure for labeling green fluorescent protein (GFP)-expressing cells in 1-micron zebrafish sections for fluorescence and electron microscope observation using a 10 nm gold-labeled secondary antibody. GFP fluorescence survived fixation in 4% paraformaldehyde/0.1% glutaraldehyde and can be visualized directly by fluorescence microscopy. For electron microscopy, thin sections of silvergold color were collected on nickel grids and were incubated in anti-GFP antibody at a 1:25 dilution for 2 hr at room temperature, followed by 10-nm immunogold-conjugated goat anti-rabbit IgG for 1 hr. After contrasting with 3% aqueous uranyl acetate solution, the grids were dried and viewed with a Hitachi 600 transmission electron microscope operated at 75 kV.
Reference:
LubyPhelps, K.; Ning, G.; Fogerty, J., and Besharse, J. C.: Visualization of Identified GFP-expressing Cells by Light and Electron Microscopy. J. Histochem. Cytochem., 51, 271-274 (2003).
Abstract (courtesy of the Journal of Histochemistry and Cytochemistry):
http://www.jhc.org/cgi/content/abstract/51/3/271
In the same issue, Jockusch, Voight and Eberhard describe a novel vapor fixation procedure for GFP-containing specimens: cryo-stat sections were dried for about 5 min and then exposed, in a tightly closed plastic dish, for 212 hr at 20C to the vapor of filter paper soaked with 37% formaldehyde. This produced superior fixation to liquid methods.
Reference:
Jockusch, H.; Voigt, S., and Eberhard, D: Localization of GFP in Frozen Sections from Unfixed Mouse Tissues: Immobilization of a Highly Soluble Marker Protein by Formaldehyde Vapor. J. Histochem. Cytochem., 51, 401-404 (2003).
Abstract (courtesy of the Journal of Histochemistry and Cytochemistry):
http://www.jhc.org/cgi/content/abstract/51/3/401
Nanogold® conjugates are also effective for localizing fluorescent proteins, and their smaller size gives them superior sample penetration, as Polischuk and co-workers demonstrate in their studies of the Golgi apparatus. Quantitative fluorescence imaging techniques and ultrastructural analysis were used to address whether the Golgi apparatus is a steady-state or a stable organelle: all classes of Golgi components were found to be dynamically associated with this organelle, contrary to the prediction of the stable organelle model. To localize p58GFP, Sec13YFP, and hemagglutinin (HA)-tagged mutant targets for immunoelectron microscopy, transfected cells were fixed and incubated with anti-GFP or anti-HA antibodies, then labeled with Nanogold-Fab fragments of secondary antibodies and developed with the GoldEnhance kit.
References:
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).
Polishchuk, R. S.; Polishchuk, E. V.; Marra, P.; Alberti, S.; Buccione, R.; Luini, A., and Mironov., A. A.: Correlative light-electron microscopy reveals the tubular-saccular ultrastructure of carriers operating between Golgi apparatus and plasma membrane. J. Cell Biol., 148, 4558 (2000).
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