Updated: September 12, 2001
N A N O P R O B E S E - N E W S
Vol. 2, No. 8 September 12, 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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Two new research applications have been introduced on our web site, describing different approaches to the organization of gold clusters into larger, supramolecular arrays. In the first, DNA strands are used as templates to deposit positively charged Nanogold® particles; these may then be linked to form molecular wires by the autometallographic deposition of silver (silver enhancement) or gold (gold enhancement):
Our paper: www.nanoprobes.com/MSADNA01.html
Charged Nanogold®: www.nanoprobes.com/LabRgts.html#charged
Gold enhancement: www.nanoprobes.com/GoldEnhance.html
In the second paper, from Microscopy and Microanalysis 2000, we describe the formation and Scanning Transmission Electron Microscope observation of proto-crystalline arrays of a gold cluster compound, dubbed "Greengold," which is similar in size to Nanogold®:
Our paper: www.nanoprobes.com/MSAXTALS00.html
See other applications, and abstracts presented by Nanoprobes scientists at past Microscopy and Microanalysis meetings, on our Applications page:
www.nanoprobes.com/Applic.html
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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: www.nanoprobes.com/TechNGlr.html#isolate
Guide - Product Isolation: www.nanoprobes.com/LGuide3.html
References - Nanogold® reagents: www.nanoprobes.com/Refnglr.html
Nanogold® references by application: www.nanoprobes.com/RefTopNG.html
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Because it is covalently linked, Nanogold® can be used to label many molecules which are not effectively labeled by colloidal gold. In our special report on Nanogold®-peptides, we describe three investigators who used peptides as short as 11 amino acids to study cellular features and processes. Use Monomaleimido Nanogold® to label peptides with cysteine residues, or Mono-Sulfo-NHS-Nanogold® to label any peptide at the N-terminal amine.
If you are working with an oligonucleotide, a reactive group, such as a thiol or an amine, may be inserted during oligonucleotide synthesis, or attached afterwards; you can then label the functionalized oligonucleotide with Nanogold® labeling reagents in the same manner as an antibody or protein. See how this process works in Alivisatos et al., Nature, 382, p. 609 (1996).
Special report - Nanogold® Peptide labeling: www.nanoprobes.com/Peptide.html
Nanogold® labeling reagents - catalog: www.nanoprobes.com/LabRgts.html
Technical Help - Nanogold® reagents: www.nanoprobes.com/TechNGlr.html
Alternative strategies for DNA labeling: www.nanoprobes.com/TechNGlr.html#DNA
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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:
Hacker, G. W., Hauser-Kronberger, C., Zehbe, I., Su, H., Schiechl, A., Dietze, O., and Tubbs, R.: In Situ localization of DNA and RNA sequences: Super-sensitive In Situ hybridization using Streptavidin-Nanogold®-Silver Staining: Minireview, Protocols and Possible Applications. Cell Vision, 4, 54-65 (1997).
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).
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: www.nanoprobes.com/InSitu.html
References for Nanogold labeling: www.nanoprobes.com/RefTopNG.html
Catalog information on Nanogold conjugates: 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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Gold-labeled DNA has been used for localization of target sequences within monolayers of oligonucleotides adsorbed to gold substrates. Csaki and co-workers used gold-conjugated oligonucleotides which were complementary to the target sequence; after hybridization, these were detected by AFM. The detection of single particles using this method gives lateral resolution in the nanometer range. Reference:
Csaki, A.; Moller, R.; Straube, W.; Kohler, J. M., and Fritzsche, W.; DNA monolayer on gold substrates characterized by nanoparticle labeling and scanning force microscopy. Nucleic Acids Res., 29, E81 (2001).
Abstract:
http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=11504889&dopt=Abstract
Two recent publications describe the simultaneous localization of nucleic acids and proteins in the same sample. Tubbs and co-workers report a three-color fluorescence method for paraffin-embedded tissue sections, in which amplification of the Her-2/neu protooncogene is detected by fluorescence in situ hybridization, nuclearmaterial is stained with DAPI, and the overexpressed oncoprotein is detected using the alkaline phosphatase chromogen nuclear fast red. Reference:
Tubbs, R. R.; Pettay, J.; Roche, P.; Stoler, M. H.; Jenkins, R.; Myles, J., and Grogan, T.; Concomitant Oncoprotein Detection with Fluorescence in Situ Hybridization (CODFISH); J. Mol. Diagn., 2, 78 (2000).
Abstract:
http://jmd.amjpathol.org/cgi/content/abstract/2/2/78
In the latest issue of the Journal of Histochemistry and Cytochemistry, Nagaso and co-workers simultaneously visualized RNA and protein for both light and fluorescent microscope observation. For fluorescent observation, digoxigenin-labeled RNA probes were visualized with alkaline phosphatase-conjugated anti-digoxigenin antibodies and nuclear fast red, while protein was visualized using Cy5-labeled secondary antibodies; for light microscopy, RNA transcripts hybridized with the digoxigenin-labeled probe were located using alkaline phosphatase-labeled anti-digoxigenin antibodies with NBT-BCIP to give a blue stain while target proteins were stained with HRP-secondary antibodies with DAB. Reference:
Nagaso, H.; Murata, T.; Day, N., and Yokoyama, K. K.: Simultaneous Detection of RNA and Protein by In Situ Hybridization and Immunological Staining. J. Histochem. Cytochem., 49, 1177 (2001).
Abstract:
http://www.jhc.org/cgi/content/abstract/49/9/1177
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