Updated: July 8, 2002
N A N O P R O B E S E - N E W S
Vol. 3, No. 7 July 9, 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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Undecagold is an 11-atom gold cluster, smaller than Nanogold$#174; but with the same covalent mode of activation and cross-linking. Although generally we recommend that you use Nanogold rather than undecagold for electron microscopy immunolabeling, undecagold has some useful applications as well. Milligan, Al-Bassam and their co-workers used mutated inter-repeat/microtubule-associated protein, cIR-MAP2/c, with a cysteine engineered into the second IR region (cys364) labeled with maleimido undecagold, to determine the location of MAP2 and tau protein binding on the surface of microtubule protofilaments. Cryo-EM and helical image analysis showed that both the IR and MAP2 elements lie along the exterior ridges of microtubules, implying that they form an ordered structure that stabilizes the microtubules.
Reference:
Al-Bassam, J.; Ozer, R. S.; Safer, D.; Halpain, S., and Milligan, R. A.: MAP2 and tau bind longitudinally along the outer ridges of microtubule protofilaments. J. Cell Biol., 157, 1187-96 (2002).
Abstract (courtesy of the Journal of Cell Biology):
http://www.jcb.org/cgi/content/abstract/157/7/1187
Undecagold is a useful label if you need the highest possible resolution, and have an instrument capable of detecting it. Use it for:
- STEM microscopy
- Image analysis
- Diffraction studies
- Heavy atom derivatization of large proteins and membrane proteins in crystallography
- As a high-resolution microscopy size standard
General and catalog information and applications: www.nanoprobes.com/UndecaG.html
References, sorted by application: www.nanoprobes.com/RefTopUG.html
Technical help: www.nanoprobes.com/TechUG.html
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Wondering how your gold labeling reaction went, and what the conjugate should look like? Our Guide to Gold Cluster labeling includes a detailed procedure for calculating labeling using UV/visible absorption data, as well as examples of the spectra of Nanogold$#174;, undecagold, and their conjugates.
The Guide also includes tips on optimizing the reaction itself, as well as the best methods for separating conjugates:
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Our attention was drawn to a recent report by Liu and co-workers, who describe the use of gold nanoparticles as amplifiers for DNA hybridization detection using a microgravimetric quartz crystal microbalance; thiolated nucleotides were coordinated to the 20 nm colloidal gold particles, then these constructs used to detect the target DNA hybridized to an initial set of oligonucleotide-modified gold particles anchored to the quartz surface in the preceding step. However, they wrongly describe these particles as "nanogold." Please remember - only the 1.4 nm covalently linked gold label made by Nanoprobes is called Nanogold$#174; - and it is trademarked!
Reference:
Liu, T.; Tang, J., and Jiang, L.: Sensitivity enhancement of DNA sensors by nanogold surface modification. Biochem. Biophys. Res. Commun., 295, 14-16 (2002).
Abstract (courtesy of Biochemical and Biophysical Research Communications):
http://www.idealibrary.com/links/doi/10.1016/S0006-291X(02)00628-9
Nanogold$#174; may be used to label many molecules, including DNA, and has opened up novel applications. Dubertret et al used Nanogold as a quencher to label molecular beacons, 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. Nanogold has been found 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; Nanoprobes has now received NIH support to investigate this effect further.
Reference:
Dubertret, B., Calame, M., and Libchaber, A.: Single-mismatch detection using gold-quenched fluorescent oligonucleotides. Nat. Biotechnol., 19, 365-370 (2001).
Abstract (Medline):
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=11283596&dopt=Abstract
Hamad-Schifferli and co-workers at the Massachusetts Institute of Technology Media Lab have demonstrated that the highly localized temperature rise produced by a conjugated Nanogold$#174; particle inductively couple to a pulsed radio frequency magnetic field may be used for the remote control of DNA hybridization. Mono-Sulfo-NHS Nanogold was used to label molecular beacons with a primary amino- group synthetically inserted into the loop. The effect was highly localized, and neighboring molecules lacking the Nanogold antenna were unaffected. The effect is fully reversible.
Reference:
Hamad-Schifferli, K.; Schwartz, J. J.; Santos, A. T.; Zhang, S., and Jacobson, J. M.: Remote electronic control of DNA hybridization through inductive coupling to an attached metal nanocrystal antenna. Nature,, 415, 152-155 (2002).
Abstract (Medline):
http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=11805829&dopt=Abstract
More information about the conjugation of Nanogold to oligonucleotides:
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We will be presenting results from several of our ongoing research projects at scientific meetings this summer. Jim Hainfeld will be chairing a symposium on the 'Use of Gold and Gold/Fluorescence for Improved Histology' at the 2002 Joint Meeting of The Japan Society of Histochemistry and Cytochemistry and The Histochemical Society (July 18 - 21, Seattle, WA) which will include results from ourselves and our collaborators; this session will be from 2 to 5 pm on Thursday, July 18. Meanwhile, in the JSHC Workshop on 'Special technologies in histochemistry,' Dr. Raymond Tubbs will give a presentation on in situ hybridization detection methods, including Nanogold$#174; with autometallography.
We will also be giving three presentations at Microscopy and Microanalysis 2002 (the Microscopy Society of America Annual Meeting) in Quebec City, August 4 - 8, 2002. We will give a poster presentation of some results obtained with our prototype ALEXA FluoroNanogold conjugates at 1:00 pm on Tuesday, August 6 (Poster # 167, Program book #403), and a poster presentation on our novel enzyme metallographic detection method at 1:00 pm on Wednesday, August 7 (Poster # 228, Program book #664). In addition, we will be presenting new results obtained with our prototype nitrilotriacetic acid-Ni(II) gold cluster reagents in the symposium on 3-D electron microscopy of macromolecules, on Tuesday August 6 at 4:00 pm in Room 301B (Program book #451).
Meeting information: http://www.microscopy.com/MSAMeetings/MMMeeting.html
Nanoprobes has also received a new Phase 2 Small Business Innovation Research (SBIR) grant from the National Institute of General Medical Sciences (NIH) of $ 986,617 for two years, effective July 1, to develop gold quenched molecular beacons. This technology will simplify and enhance the real-time detection of specific DNA sequences in cells and other highly localized systems. This technology may help enable applications such as real-time PCR-based cancer screening, fiber optic biosensors for quickly screening tissues during transplantation and other medical procedures, and portable fetal testing.
More details: www.nanoprobes.com/News02.html#2R44GM62100
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Briane and Group have described an immunogold procedure used to trace the cellular route of cationic liposomes in tumor MCF7 cells by transmission electron microscopy. Cationic liposomes prepared from TMAEC-Chol [3 beta-(N-(N',N',N'-trimethylaminoethane)-carbamoyl) cholesterol iodide] and TEAPC-Chol [3 beta-(N-(N',N',N'-triethylaminopropane)-carbamoyl) cholesterol iodide], two cholesterol-based cationic lipids containing a quaternary ammonium. Anti-digoxigenin Fab fragments labeled with 1 nm gold were used to localize liposomally encapsulated, digoxigenin-labeled DNA plasmids. Early after the beginning of transfection (30 min, 1 hr, 5 hr), gold particles were observed only in the cytoplasm and in endosome-like vesicles, whereas after 24 hr gold particles were densely present in the nucleus, demonstrating the nuclear localization of plasmids vectorized by the cationic liposomes used.
Reference:
Briane, D.; Lesage, D.; Cao, A.; Coudert, R.; Lievre, N.; Salzmann, J. L., and Taillandier, E.: Cellular Pathway of Plasmids Vectorized by Cholesterol-based Cationic Liposomes. J. Histochem. Cytochem., 50, 983-92 (2002).
Abstract (courtesy of the Journal of Histochemistry and Cytochemistry):
http://www.jhc.org/cgi/content/abstract/50/7/983
In the same issue, Kurabuchi and Tanaka used a two-sided double immunogold labeling technique to stain sections of mouse thyroid and respiratory tract using biotinylated primary antibodies and streptavidin conjugated to different sized gold particles to show that localization of PC1 and PC2 was restricted to the calcitonin/CGRP-producing parafollicular cells in the thyroid; in the respiratory tract, only PC1 immunoreactivity was observed, and a subpopulation of PC1-immunoreactive cells contained CGRP. The authors inferred that procalcitonin is proteolytically cleaved by PC2 alone or by PC2 together with PC1, and that the proCGRP is cleaved by PC1.
Reference:
Kurabuchi, S., and Tanaka, S.: Optimization of immunogold labeling TEM: Immunocytochemical Localization of Prohormone Convertases PC1 and PC2 in the Mouse Thyroid Gland and Respiratory Tract. J. Histochem. Cytochem., 50, 903-910 (2002).
Abstract (courtesy of the Journal of Histochemistry and Cytochemistry):
http://www.jhc.org/cgi/content/abstract/50/6/863
Rindi and co-workers used a double immunogold labeling protocol with 10 and 20 nm gold to study the developmental biology of the production of Ghrelin, a new gastric peptide; their findings confirmed that ghrelin cells are a distinct type relative to histamine enterochromaffin-like, somatostatin D, glucagon A or serotonin enterochromaffin cells.
Reference:
Rindi, G.; Necchi, V.; Savio, A.; Torsello, A.; Zoli, M.; Locatelli, V.; Raimondo, F.; Cocchi, D., and Solcia, E.: Characterisation of gastric ghrelin cells in man and other mammals: studies in adult and fetal tissues. Histochem. Cell Biol., 117, 511519 (2002).
Abstract (courtesy of Histochemistry and Cell Biology):
http://link.springer.de/link/service/journals/00418/contents/02/00415/
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