Updated: March 6, 2002
N A N O P R O B E S E - N E W S
Vol. 3, No. 3 March 6, 2002
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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Akaki and co-workers recently used negatively charged Nanogold® to confirm the distribution of electrical charges over the surface of Plasmodium falciparum merozoites and erythrocytes. They used atomic force microscopy with surface potential spectroscopy to map the surface charge directly, then followed up with transmission electron microscopy. Negatively charged Nanogold was incubated with merozoites at pH 7.4 for 30-40 seconds, then after washing the gold particles were enlarged with silver enhancement and gold toning for TEM observation; cationic ferritin was used to label the positively charged regions of erythrocytes. They found that the apical ends of merozoites were labeled with negatively charged Nanogold, while the entire erythrocyte surface was labeled with cationic ferritin, and inferred that the surface charge at the apical end of the merozoite may play a role in the invasion of the erythrocyte. Reference:
Akaki, M.; Nagayasu, E.; Nakano, Y., and Aikawa, M.: Surface charge of Plasmodium falciparum merozoites as revealed by atomic force microscopy with surface potential spectroscopy. Parasitol. Res., 88, 16-20 (2002).
Abstract (Medline):
http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=11822732&dopt=Abstract
Positively charged Nanogold®, at neutral or low pH, ionizes to give a positively charged species. Prescianotto-Baschong and co-workers found that positively charged Nanogold is a useful marker for the endocytic pathway in yeast. During a time course of incubation of yeast spheroplasts with positively charged Nanogold at 15C, the gold was detected sequentially in small vesicles, a peripheral, vesicular/tubular compartment designated as an early endosome, a multivesicular body corresponding to the late endosome near the vacuole, and in the vacuole. Reference:
Prescianotto-Baschong, C., and Riezman, H.: Morphology of the yeast endocytic pathway. Mol. Biol. Cell, 9, 173-89 (1998).
Abstract (Molecular Biology of the Cell):
http://www.molbiolcell.org/cgi/content/abstract/9/1/173
Reprint (PDF):
http://www.molbiolcell.org/cgi/reprint/9/1/173.pdf
The affinity of the positively charged gold for negatively charged macromolecules may be used as an organizing mechanism for the formation of molecular wires: as an example, DNA strands were used as templates to deposit positively charged Nanogold particles, which may then be linked to form conductive molecular wires by the autometallographic deposition of silver (silver enhancement) or gold (gold enhancement). This is described in our extended abstract from Microscopy and Microanalysis 2001:
Our paper: www.nanoprobes.com/MSADNA01.html
Gold enhancement: www.nanoprobes.com/GoldEnhance.html
More information:
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Is background staining or over-development a problem when you use gold labeling with silver or gold enhancement? Try these suggestions to reduce it:
- For HQ Silver; try washing with 0.02 M sodium citrate buffer, pH 7.0, before silver enhancement (Reference: 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)).
- For GoldEnhance: wash with freshly prepared 2.5 % sodium thiosulfate for 1 minute after GoldEnhance development.
- Lugol's iodine (Sigma) - this reagent is used to remove heavy metals from tissue slides for light microscopy, which eliminates one source of background. We have found it to be essential for in situ hybridization detection with Nanogold® and GoldEnhance; it is applied early, before the Nanogold reagent.
- Wash with 0.6 M triethylammonium bicarbonate buffer in 20 % isopropanol/water after gold enhancement, or before silver enhancement. This buffer is highly effective for dissolving and removing unattached small gold particles such as Nanogold. It is prepared by bubbling carbon dioxide through a mixture of degassed water and degassed triethylamine; we usually make a 2 M stock solution, then dilute in water/isopropanol to give 0.6 M in 20 % isopropanol/water (Reference: Safer, D.; Bolinger, L., and Leigh, J. S.; J. Inorg. Biochem., 26, 77 (1986)).
- If your system can tolerate it, wash with 20 % DMSO in water after gold enhancement or before silver enhancement. DMSO is also an excellent solvent for Nanogold and may be effective for the removal of other small gold particles.
If you have other technical questions, check our technical help resources:
For references and descriptions of protocols that our users have developed with Nanogold, check our review articles in the Journal of Histochemistry and Cytochemistry (Hainfeld, J. F., and Powell, R. D.: J. Histochem. Cytochem., 48, 471-480 (2000)), or Cell Vision (Hainfeld, J. F., and Powell, R. D.: Cell Vision, 4, 408-432 (1997)).
E-mail us (tech@nanoprobes.com) if you would like a copy of the Cell Vision paper.
Abstract of article from the Journal of Histochemistry and Cytochemistry:
http://www.jhc.org/cgi/content/abstract/48/4/471
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Because it is covalently attached, Nanogold® may be used to label many molecules which cannot be labeled with colloidal gold, and this has opened novel applications. Dubertret et al have 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. 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 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.
More information: http://www.histochemicalsociety.org/meetings/index2002.html
Also, look for our presentations at Microscopy and Microanalysis 2002 (the Microscopy Society of America Annual Meeting) in Quebec City, August 4 - 8, 2002.
More information: http://www.microscopy.com/MSAMeetings/MMMeeting.html
If you are reading this from Taiwan, you can order our products from a new distributor, AWTC, Inc. You can contact them at:
AWTC, INC.
Floor 4, No. 14, Hoping East Road
Section 1
Taipei 106
Taiwan
Tel: (886) 2-23681696
Fax: (886) 2-23684339
E-mail: sanjo@ms3.hinet.net
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Pandey and co-workers correlate immunogold labeling showing the cellular distribution with western blotting and protein expression (determined by radioiodination) to show that serotonin 5-HT2A receptors are highly expressed in the brains of teenage suicide victims. Reference:
Pandey, G. N.; Dwivedi, Y.; Rizavi, H. S.; Ren, X.; Pandey, S. C.; Pesold, C. Roberts, R. C,; Conley, R. R., and Tamminga C. A.: Higher Expression of Serotonin 5-HT(2A) Receptors in the Postmortem Brains of Teenage Suicide Victims. Am. J. Psychiatry, 159, 419-29 (2002).
Abstract (American Journal of Psychiatry):
http://ajp.psychiatryonline.org/cgi/content/abstract/159/3/419
Neumann and Gabel, meanwhile, report on a simple method for reducing autofluorescence in aldehyde-fixed neural tissue: irradiate with light before treatment with fluorescence probes. Reference:
Neumann, M., and Gabel, D.: Simple Method for Reduction of Autofluorescence in Fluorescence Microscopy. J. Histochem. Cytochem., 50, 437-439 (2002) .
Abstract (JHC Online):
http://www.jhc.org/cgi/content/abstract/50/3/437
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