Updated: December 10, 2001

N A N O P R O B E S E - N E W S

Vol. 2, No. 11 December 10, 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.

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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Nanogold and Signal Amplification for Low Abundance Targets

If you are concerned about low abundance of your target, or have experienced low labeling, whether nucleic acid or antigen, you can increase the labeling by using a signal amplification procedure such as NEN's Tyramide Signal Amplification process. This has been highly effective for in situ hybridization detection:

Zehbe, I., Hacker, G.W., Su, H., Hauser-Kronberger, C., Hainfeld, J.F., and Tubbs, R.: Am. J. Pathol. 150, 1553-1561 (1997).
Abstract: http://ajp.amjpathol.org/cgi/content/abstract/150/5/1553

However, you can also use this method to amplify cellular antigens:

Weipoltshammer, K.; Schofer, C.; Almeder, M., and Wachtler, F.: Signal enhancement at the electron microscopic level using Nanogold and gold-based autometallography. Histochem. Cell Biol., 114, 489-495 (2000).
Abstract: http://link.springer-ny.com/link/service/journals/00418/bibs/0114006/01140489.htm

Nanogold conjugates can also be used to detect products amplified by PCR:

Hacker, G. W.; Zehbe, I.; Hainfeld, J.; Sällström, J.; Hauser-Kronberger, C.; Graf, A.-H.; Su, H.; Dietze, O., and Bagasra, O; High-Performance Nanogold® In Situ Hybridization and In Situ PCR. Cell Vision, 3, 209 (1996).

For more information about the use of Nanogold in these applications:

Application - In situ hybridization: http://www.nanoprobes.com/InSitu.html
References for Nanogold labeling: http://www.nanoprobes.com/RefTopNG.html
Catalog information on Nanogold conjugates: http://www.nanoprobes.com/NanoAb.html

Tyramide Signal Amplification (TSA®), or Catalyzed reporter deposition (CARD), was developed by Dr. Mark Bobrow et al. (J. Immunol. Meth. 1989;124:279-85), is patented by NEN® Life Sciences Products (Boston, MA, USA; Web: http://www.nenlifesci.com).

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Make Molecular Wires or Label Endocytotic Components with Positively Charged Nanogold

Positively charged Nanogold® contains multiple amino groups, and at neutral or low pH ranges it ionizes to give a positively charged species with useful properties. The affinity of the positively charged gold for negatively charged macromolecules may be used as an organizing mechanism for the formation of molecular wires. Recently, DNA strands were used as templates to deposit positively charged Nanogold particles; these may then be linked to form conductive molecular wires by the autometallographic deposition of silver (silver enhancement) or gold (gold enhancement). For more information, see our extended abstract on DNA Nanowires from Microscopy and Microanalysis 2001:

Our paper: http://www.nanoprobes.com/MSADNA01.html
Gold enhancement: http://www.nanoprobes.com/GoldEnhance.html

Positively charged Nanogold also functions as an effective marker for the endocytic pathway in yeast. Prescianotto-Baschong and co-workers found that positively charged Nanogold binds extensively to the surface of yeast spheroplasts, and was internalized in an energy-dependent manner. During a time course of incubation of yeast spheroplasts with positively charged Nanogold at 15°C, the gold was detected sequentially in small vesicles, a peripheral, vesicular/tubular compartment that we designate 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.: Mol. Biol. Cell, 9, 173-89 (1998).
Reprint: http://www.molbiolcell.org/cgi/reprint/9/1/173.pdf

More information:

Catalog information: http://www.nanoprobes.com/LabRgts.html#charged
Product information: http://www.nanoprobes.com/Inf2022.html
Other product and research applications: http://www.nanoprobes.com/Applic.html

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Isolation of Nanogold-Labeled Conjugates

Because Nanogold® is so uniform in size, we recommend gel filtration as the method of choice for separating Nanogold conjugates from either excess Nanogold or from unlabeled biomolecules. We have expanded our technical help page for Nanogold labeling reagents to include a section on planning your labeling reaction for the easiest separation of the labeled product. In addition, separation methods and media are also discussed in our Guide to Gold Cluster Labeling:

Technical Help: conjugate isolation: http://www.nanoprobes.com/TechNGlr.html#isolate
Guide: Product Isolation: http://www.nanoprobes.com/LGuide3.html
References: Nanogold reagents: http://www.nanoprobes.com/Refnglr.html
Nanogold references by application http://www.nanoprobes.com/RefTopNG.html

The simplest way to reduce the reaction mixture to a volume suitable for injection onto a gel filtration column (5 % of column volume or smaller) is to use a centrifuge membrane concentrator with a molecular weight cut-off below the molecule you are labeling, such as the Centricon (Millipore).

Guide to Gold Cluster Labeling: http://www.nanoprobes.com/LGuide.html

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Gold Lipids: Localize Liposomes by Light and Electron Microscopy

Nanogold-labeled lipids are a unique probe from Nanoprobes, containing the 1.4 nm Nanogold cluster chemically linked to palmitoyl or dipalmitoyl phosphatidyl ethanolamine moieties. These provide a unique method for gold-labeling liposomes for either light or electron microscope observation. This method has been used to follow the liposomal delivery of an anti-fungal channel-forming drug; Nanogold-labeled liposomes containing the drug were shown to enter the cytoplasm, while those without the drug did not penetrate the cell wall. Reference:

Adler-Moore, Bone Marrow Transplantation, 14, S3-S7 (1994)).
Abstract:
http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=7703928&dopt=Abstract

Labeling with DPPE-Nanogold has also been used to demonstrate the targeting of cationic liposomes to endothelial cells in tumors and chronic inflammation. Reference:

Thurston, G., McLean, J. W., Rizen, M., Baluk, P., Haskell, A., Murphy, T. J., Hanahan, D., and McDonald, D. M.: J. Clin. Invest., 101, 1401-1413 (1998).
Reprint: http://www.jci.org/cgi/reprint/101/7/1401.pdf

See our MSA paper (http://www.nanoprobes.com/MSA96lip.html).
About Gold Lipids: http://www.nanoprobes.com/GoldLip.html
Product information - DPPE-Nanogold: http://www.nanoprobes.com/Inf4021.html
Complete list of references: http://www.nanoprobes.com/RefLip.html

Our paper:

Hainfeld, J. F., et al: Metallosomes. J. Struct. Biol., 127, 152-160 (1999).
Abstract: http://www.idealibrary.com/links/artid/jsbi.1999.4145/production.

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Recent Publications

Sakata and co-workers used Nanogold labeling in their studies of the transport of apolipoprotein B (apoB) between the endoplasmic reticulum (ER) and Golgi in HepG2 cells; they used immunoelectron microscopy with Nanogold-Fab' and HQ Silver enhancement on digitonin-permeabilized cells to localize proteasomes in close proximity to the cytosolic side of the ER membrane, concluding that newly synthesized apoB is localized throughout the entire ER and degraded homogeneously, most likely by neighboring proteasomes located on the cytosolic side of the ER membrane.

Reference:
Sakata, N.; Phillips, T. E., and Dixon, J. L.: Distribution, transport, and degradation of apolipoprotein B-100 in HepG2 cells. J. Lipid Res., 42, 1947 (2001).

Abstract:
http://www.jlr.org/cgi/content/abstract/42/12/1947

Reiner and co-workers describe double labeling with 5 and 10 nm colloidal gold to demonstrate the colocalization of caveolin-1 and endothelial NOS in myocardial capillary endothelium at the subcellular level in the rat heart, and to show the perturbation of this pattern by bradykinin stimulation.

Reference:
Reiner, M.; Bloch, W., and Addicks, K: Functional Interaction of Caveolin-1 and eNOS in Myocardial Capillary Endothelium Revealed by Immunoelectron Microscopy. J. Histochem. Cytochem., 49, 1605-1610 (2001).

Abstract:
http://www.jhc.org/cgi/content/abstract/49/12/1605

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