N A N O P R O B E S     E - N E W S

Vol. 3, No. 9          September 12, 2002

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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New Research Applications: NTA-Ni(II) Gold and Enzymatic Metallography

Nitrilotriacetic acid (NTA) is a tricarboxylic acid, monoamino chelator that forms a stable complex with nickel (II). This leaves two vacant coordination sites on the nickel (II) ion; through these, the complex binds strongly to polyhistidine sequences. Nanoprobes has attached the NTA-Ni(II) chelate to gold labels to give a new type of probe, which is used to localize and detect polyhistidine-tagged proteins. This new labeling reagent is much smaller even than antibody Fab' fragments, and can be used directly to localize any target which can incorporate a polyhistidine tag. Some results obtained using prototypes are described in our abstract from Microscopy and Microanalysis 2002:

http://www.nanoprobes.com/MSANTA02.html

Other references have described results obtained using prototypes of this probe, and we plan to introduce it as a commercial product in the next few months. When it is introduced, it will be announced on our web site.

Other references: http://www.nanoprobes.com/RefTopNG.html#nta
More information: http://www.nanoprobes.com/LGuide5.html#func

Enzymatic metallography refers to a new staining method underdevelopment by Nanoprobes, in which an enzyme conjugate targeted to a site of interest selectively deposits metal from solution. This results in a dense, black, highly localized signal, which may be used for immunohistochemical staining and in situ hybridization in the brightfield light microscope. See some results obtained with this procedure in our abstract from Microscopy and Microanalysis 2002:

http://www.nanoprobes.com/MSAENZ02.html

More information and applications of our products:

• Applications index: http://www.nanoprobes.com/Applic.html
• References by product or application: http://www.nanoprobes.com/Ref.html

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Colloidal Gold and Covalent Gold

If you are looking for help with your colloidal gold conjugation, Research Diagnostics, Incorporated include a useful procedure for antibody conjugation on their web site:

http://www.researchd.com/gold/gold8.htm

At Nanoprobes, we are working to bring some of the advantages of Nanogold® - site-specific linking, conjugation to novel probes, preparation of chemically selective labeling reagents - to larger gold probes. See some preliminary results using a covalently linked 10 nm gold-Fab' conjugate in our abstract from Microscopy and Microanalysis 1999:

http://www.nanoprobes.com/MSALG99.html

Find out about the use of gold probes for lateral flow devices and other medical diagnostics, 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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Combined ALEXA-488 and Nanogold® Probes

In our program to expand our line of FluoroNanogold reagents, we are currently developing antibody and streptavidin conjugates labeled with ALEXA-488 (from Molecular Probes) and Nanogold. Compared with conventional fluorescein probes, these offer much brighter, less pH-dependent fluorescence with lower photobleaching. These new reagents will be available in the next few months from Nanoprobes and from Molecular Probes; you can learn more about these new probes and see some results in our abstract from Microscopy and Microanalysis 2002:

http://www.nanoprobes.com/MSAFN02.html

One issue which can arise with these probes is controlling non-specific background binding. FluoroNanogold is susceptible to this because it combines two labels that are frequently used in different concentrations under different conditions. Compromise and optimization may be required to obtain the best performance.

The following methods can help to reduce background:

• Blocking with 5 % nonfat dried milk. This was found to be particularly effective when mixed with and added to the specimen with the FluoroNanogold conjugate. Cold-water fish gelatin may also be helpful.

• Manual control of camera exposure. If FluoroNanogold is compared with larger, more highly labeled fluorescently conjugated IgG conjugates with brighter fluorescence, and automatic exposure adjustment allowed with FluoroNanogold-stained specimens, the greater exposure can lead to higher apparent backgrounds. Setting the camera exposure manually can be used to overcome this effect.

• Sodium citrate buffer wash after FluoroNanogold application but before silver enhancement. 0.02 M sodium citrate at pH 7.0 works well with HQ Silver, while pH 3.5 works best with the Danscher silver formulation.

• Reducing hydrophobic interactions. Both the gold and fluorescent labels have some hydrophobicity: therefore, including in the wash buffer agents that reduce hydrophobic interactions may help to reduce non-specific binding. Suitable reagents:
  • 0.6 M triethylammonium bicarbonate buffer (prepared by bubbling carbon dioxide into an aqueous suspension of triethylamine with stirring: see Safer, D.; Bolinger, L., and Leigh, J. S.; J. Inorg. Biochem., 26, 77 (1986));
  • 0.1 % to 1 % detergent, such as Tween-20, or Triton X-100;
  • 0.1 % to 0.5 % of an amphiphile, such as benzamidine or 1,2,3-trihydroxyheptane.

• Catalog information: http://www.nanoprobes.com/FluoroN.html
• Technical help for FluoroNanogold: http://www.nanoprobes.com/TechFN.html
• References for FluoroNanogold: http://www.nanoprobes.com/RefTopFN.html
• Molecular Probes: http://www.probes.com

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Test Sites Wanted: Correlative Microscopy with NTA-Gold and GFP

Are you using Green Fluorescent Protein, and interested in correlative fluorescent and electron microscopic imaging in living cells? If you can generate a polyhistidine-tagged GFP fusion protein, we would be very interested in localizing it using nitrilotriacetic acid (NTA)-Ni(II) gold, as a new approach to combined fluorescent and gold labeling. If you can make such a probe and would like to try this, please let us know.

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Nanoprobes Receives New Grants

Press release: http://www.nanoprobes.com/News02.html#1R43CA094495

The second is a Phase 2 Small Business Technology Transfer grant in collaboration with the Cleveland Clinic Foundation to develop and clinically test a chromogenic Her-2/neu gene amplification assay. This in situ hybridization assay used Nanogold-labeled streptavidin with gold enhancement to give a black signal which is observed and interpreted using a conventional brightfield light microscope without the need for oil immersion.

Press Release: http://www.nanoprobes.com/News02.html#2R42CA83618

A prototype form of this assay and results from 100 breast cancer specimens have been published, and an interobserver reproducibility study has shown high reproducibility.

Reference - method:

Tubbs, R.; Pettay, J.; Skacel, M.; Powell, R.; Stoler, M.; Roche, P., and Hainfeld, J.: Gold-Facilitated in Situ Hybridization: A Bright-Field Autometallographic Alternative to Fluorescence in Situ Hybridization for Detection of HER-2/neu Gene Amplification. Am. J. Pathol., 160, 1589-1595 (2002).

Abstract (courtesy of the American Journal of Pathology):
http://ajp.amjpathol.org/cgi/content/abstract/160/5/1589

Reference - reproducibility study:

Tubbs, R.; Skacel, M.; Pettay, J.; Powell, R.; Myles, J.; Hicks, D.; Sreenan, J.; Roche, P.; Stoler, M. H., and Hainfeld, J.: Interobserver Interpretative Reproducibility of GOLDFISH, A First Generation Gold-Facilitated Auto-metallographic Bright Field In Situ Hybridization Assay for HER-2/neu Amplification in Invasive Mammary Carcinoma. Am. J. Surg. Pathol., 26, 908-913 (2002).

Abstract (courtesy of Lippincott, Williams and Wilkins):
http://ipsapp003.lwwonline.com/content/getfile/2302/32/9/abstract.htm

More information:

• About Nanogold-ISH detection: http://www.nanoprobes.com/InSitu.html
• Product Information - Nanogold-Streptavidin: http://www.nanoprobes.com/Inf2016.html
• Gold enhancement: http://www.nanoprobes.com/GoldEnhance.html

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Other Recent Publications

Stromer and co-workers have used double immunofluorescence labeling to identify colocalization of proteins in smooth muscles, followed by electron microscopy immunolabeling with protein-A - 12 nm gold to localize individual proteins to specific cellular components. 60-90 nm thin sections were cut from Lowicryl K4 M Unicryl resin-embedded vas deferens, porcine external iliac artery, and chicken gizzard, and mounted on Formvar-carbon-coated nickel grids for electron microscopy, and on poly-L-lysine coated coverglasses for fluorescence microscopy. The results indicate that there is extensive overlap between the locations of contractile and cytoskeletal elements and, thus, do not support the two-domain model of smooth muscle structure.

Reference:

Stromer, M. H.; Mayes, M. S., and Bellin, R. .M.: Use of actin isoform-specific antibodies to probe the domain structure in three smooth muscles. Histochem. Cell Biol., Online First publication DOI 10.1007/s00418-002-0453-8 (2002).

Abstract (courtesy of Histochemistry and Cell Biology):
http://link.springer.de/link/service/journals/00418/contents/02/00453/

JoAnne Buchanan and co-workers have briefly described a method for the electron microscope localization of Green Fluorescent Protein (GFP): use a primary antibody against GFP, followed by a Nanogold-labeled secondary antibody followed by silver enhancement. Hippocampal neurons were transfected with the PLC-d PH domain fused to GFP, which binds to phosphatidyl 4,5-biphosphate (PIP2). Microwave fixation followed by pre-embedding labeling with anti-GFP antibody (Roche) and Nanogold-labeled secondary was used to localize the GFP for electron microscope observation.

Reference:

Buchanan, J.; Micheva, K., and Smith, S. J.: Immunolocalization of GFP fusion proteins in hippocampal neurons using anti-GFP antibodies and nanogold labeling. Mol. Biol. Cell., 11 (Supplement), 277a (2000).

Branched DNA in situ hybridization is shown to be a highly sensitive method for localizing DNA by in situ hybridization in tissue samples by Kenny and co-workers. This group explored the cell type-specific distribution of HPV DNA and mRNA by bDNA ISH using normal and human papillomavirus (HPV)-infected cervical biopsy specimens. bDNA ISH allowed rapid, sensitive detection of nucleic acids with high specificity while preserving tissue morphology. This method uses hybridization with a branched-chain DNA probe as a mechanism for signal amplification; binding is visualized using alkaline phosphatase, allowing the use of nuclear fast red substrate for both brightfield and fluorescence visualization.

Reference:

Kenny, D.; Shena, L.-P., and Kolberg, J. A.: Detection of Viral Infection and Gene Expression in Clinical Tissue Specimens Using Branched DNA (bDNA) In Situ Hybridization. J. Histochem. Cytochem., 50, 1219-1227 (2002).

Abstract (courtesy of the Journal of Histochemistry and Cytochemistry):
http://www.jhc.org/cgi/content/abstract/50/9/1219

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