Updated: June 11, 2001
N A N O P R O B E S E - N E W S
Vol. 2, No. 5 June 11, 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.
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When combined with silver or gold enhancement, Nanogold labeling is also a powerful method for light microscopy:
- The signal is often more sensitive than competing detection technologies.
- Black staining is highly visible with counter-stains.
- Brightfield LM observation is more convenient than fluorescence.
- Signal is permanent; does not fade.
- Can be combined with enzymatic staining for double labeling.
Some applications:
For more references, see our complete list:
www.nanoprobes.com/RefTopNG.html#Nlight
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GoldEnhance works like silver enhancement, but instead deposits gold. Gold enhanced particles are denser than silver enhanced ones for superior back-scattered electron (BSE) detection in the scanning electron microscope (SEM). Gold enhancement is impervious to osmium etching, and also features a longer time window from full development to the appearance of background, so it is easier to use in blotting and diagnostics applications. Unlike silver enhancement reagents, GoldEnhance may be used in the presence of chloride buffers without precipitation problems. GoldEnhance also produces excellent results for light microscopy; see our references page for details.
GoldEnhance: information and prices: www.nanoprobes.com/GoldEnhance.html
Our GoldEnhance paper from M & M 99: www.nanoprobes.com/MSAGE99.html
References: www.nanoprobes.com/Refge.html
If you would prefer to use silver enhancement, you can use gold toning to avoid osmium tetroxide etching. If you are not using uranyl acetate, silver etching is rarely a problem; however, if it is, Burry and co-workers have found that it may be greatly reduced by using 0.1 % OsO4 instead of 1 %; osmication was performed for 30 minutes. This was found to give similar levels of staining to those found using 1 % osmium tetroxide, but 0.1 % OsO4 can be safely used after silver enhancement without gold toning. Reference:
Burry, R.W.:in M. A. Hayat ed. "Immunogold silver staining: Principles, methods and applications," CRC Press, Boca Raton, FL (1995).
If uranyl acetate staining is performed after osmication, the etching of silver is sometimes much worse, and the silver particles can be stripped away completely. We recommend gold toning if you intend to use osmication (at any concentration) and uranyl acetate. There are two procedures you can use:
- After silver enhancement, wash thoroughly with deionized water.
- 0.05 % gold chloride: 10 minutes at 4°C.
- Wash with deionized water.
- 0.5 % oxalic acid: 2 minutes at room temperature.
- 1 % sodium thiosulfate (freshly made) for 1 hour.
- Wash thoroughly with deionized water and embed according to usual procedure.
References:
Arai, R., et al.; Brain Res. Bull., 28, 343-345 (1992).
Medline citation: http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=1375863&dopt=Abstract
Arai, R., and Nagatsu, I.; in: "Immunogold-Silver Staining: principles, Methods and Applications;" M. A. Hayat, ed. Chap. 13, pp 209-216. CRC Press, Boca Raton. FL (1995).
Alternatively, this slightly gentler procedure has been reported:
- Rinse twice quickly in distilled water.
- 0.05 M sodium acetate (1 minute) then rinse again quickly.
- 0.05 % tetrachloroauric acid (2 minutes).
- Thorough rinsing in distilled water for 10 minutes, then osmicate.
Reference:
Sawada, H., and Esaki, J.: Elect. Micro., 43, 361-66 (1994).
Medline citation: http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=7722428&dopt=Abstract
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Want more information about the use of gold probes for lateral flow devices and other medical diagnostics? Read about this application, the types of gold probes used, and the factors that are important, in this article by John Chandler, Tracey Gurmin, and Nicola Robinson from IVD Technology magazine, March 2000:
Article: http://www.devicelink.com/ivdt/archive/00/03/004.html
IVD Technology site: http://www.devicelink.com/ivdt/
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Because Nanogold and undecagold are discrete compounds, they are highly regular in size, and can be used to probe for the size of pores or gaps in biological stuctures. For example, Schwartz and Matouschek have used Nanogold and undecagold to determine the dimensions of the protein import channels in the outer and inner mitochondrial membranes; the larger Nanogold was excluded, while the smaller undecagold was able to pass through the channels (See: Schwartz, M. P., and Matouschek, A.: Proc. Natl. Acad. Sci. USA, 96, 13086-13090 (1999)).
Abstract (html): http://www.pnas.org/cgi/content/abstract/96/23/13086
Full paper (PDF format): http://www.pnas.org/cgi/reprint/96/23/13086.pdf
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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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Schiebel, Kowal, and co-workers have used Monomaleimido Nanogold labeling, followed by on-grid gold enhancement, to study growth of amyloid fibers formed by NM, the prion-determining region of the yeast protein Sup35p. To assess the directionality of fiber growth, we genetically engineered a mutant of NM so that it contained an accessible cysteine residue that was easily labeled after fiber formation. The mutant protein, NMK184C, were labeled with Monomaleimido Nanogold and enhanced with GoldEnhance to highly regular 6 nm particles for TEM observation. The modified protein assembled in vitro with kinetics indistinguishable from those of the wild-type protein and propagated the heritable genetic trait [PSI(+)] with the same fidelity. In reactions nucleated with prelabeled fibers, unlabeled protein assembled at both ends, showing that NM fiber growth is bidirectional.
Scheibel, T.; Kowal, A. S.; Bloom, J. D.; Lindquist, S. L.: Bidirectional amyloid fiber growth for a yeast prion determinant. Curr. Biol., 11, 366-9 (2001).
Abstract: http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=11267875&dopt=Abstract
Robinson, Takizawa and Vandr compare gold cluster labeling using Nanogold with colloidal gold labeling; they illustrate the relationship between gold probe size and labeling density (the smaller the gold, the more densely the targets are labeled) by reference to a wide variety of systems, and describe the application of both Nanogold and FluoroNanogold to a number of microscopic techniques.
Robinson, J. M.; Takizawa, T., and Vandré, D. D.: Applications of gold cluster compounds in immunocytochemistry and correlative microscopy: comparison with colloidal gold. J. Microsc., 199, 163-79 (2000).
Abstract: http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=10971797&dopt=Abstract
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