Updated: February 5, 2002

N A N O P R O B E S     E - N E W S

Vol. 3, No. 2          February 5, 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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Nanogold®: the Advantages of Covalent Labeling

Calmodulin is a tightly bound, intrinsic subunit (delta) of the hexadecameric phosphorylase-b kinase holoenzyme. The recent use of Nanogold®-labeled calmodulins, which were exchanged for delta to enable the localization of the delta subunit within the bridged, bilobal phosphorylase b kinase holoenzyme complex by scanning transmission electron microscopy, demonstrates the versatility and resolution which may be achieved by site-specific, covalent Nanogold labeling:

Traxler, K. W.; Norcum, M. T.; Hainfeld, J. F., and Carlson, G. M.: Direct Visualization of the Calmodulin Subunit of Phosphorylase Kinase via Electron Microscopy Following Subunit Exchange. J. Struct. Biol., 135, 231-8 (2001).

Abstract (Academic Press):

The delta subunits were determined to be near the edge of the lobes, just distal to the interlobal bridges and proximal to a previously identified region of the enzymes catalytic gamma subunit.

Nanogold can be site-specifically conjugated to a wide variety of molecules which cannot be labeled with colloidal gold, including:

Once prepared, conjugates are stable under a wide range of conditions, and the extent of labeling can be calculated precisely from the UV/visible spectrum of the conjugate using the known extinction coefficients of the Nanogold. Labeling reagents are available with three different reactivities and cross-linking chemistries: Monomaleimido Nanogold, which reacts with thiols such as cysteine residues; Mono-Sulfo-NHS-Nanogold, which reacts with primary aliphatic amines, such as N-terminal amines or lysine residues; and Monoamino Nanogold, which may be activated and cross-linked using a variety of hetero- and homobifunctional cross-linking reagents.

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A New, Highly Sensitive Pre-Embedding Immunogold Procedure

Pre-embedding labeling for TEM observation is one of the principal applications of Nanogold® conjugates. In the Proceedings of Microscopy & Microanalysis 2001, Grondin and Beaudoin report a new pre-embedding immunogold method which gives a very high signal combined with very good ultrastructural preservation. It was demonstrated by labeling NADPase type 1 (a membrane protein) in confluent endothelial cells:
  1. Fix cells in situ for 3 hours with a freshly prepared and filtered solution of 1 % l-lysine, 4 % paraformaldehyde, 0.04 % glutaraldehyde and 0.25 % sodium metaperiodate in 0.04 M sodium cacodylate buffer, pH 7.4.
  2. Permeabilize with 50 % methanol at -20°C for 5 minutes.
  3. Wash with phosphate-buffered saline containing 0.1 % Tween-20 (PBST).
  4. Block unspecific labeling with phosphate-buffered saline containing 0.1 % Tween-20, 2 % goat serum, 1 % bovine serum albumin, 0.45 % fish gelatin, and 0.4 % glycine (PBSB).
  5. Incubate at 4°C overnight with primary antibody diluted 1 : 300 in PBSB.
  6. Wash three times in PBST.
  7. Incubate 1 hour at room temperature with Nanogold-labeled secondary antibody diluted 1 : 300 in PBSB.
  8. Wash three times in PBST.
  9. Fix 1 hour in 1.2 % glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.4, with 5 % sucrose.
  10. Wash with water.
  11. Enhance using GoldEnhance LM (6 minutes).
  12. Post-fixation in 2 % osmium tetroxide and 2 % potassium ferricyanide in the same buffer.
  13. Dehydrate, embed in Epon 812, section and stain with lead citrate before examination in the electron microscope.

Grondin, G., and Beaudoin, A. R.: A New Pre-Embedding Immunogold Method that Permits to Obtain a Very High Signal with a Very Good Ultrastructure. Microsc. Microanal., 7, (Suppl. 2: Proceedings) (Proceedings of the Fifty-Ninth Annual Meeting, Microscopy Society of America); Bailey, G. W.; Price, R. L.; Voelkl, E., and Musselman, I. H., Eds.; Springer-Verlag, New York, NY, 2001, pp. 1044-1045.

Dr. Susan Cheng and co-workers at NINDS have refined their pre-embedding procedure for the most uniform, consistent and reliable pre-embedding labeling, using Nanogold®-Fab' and HQ Silver. You can read their procedure in full in the technical support section of our web Site, or look up their article. References:

Tanner, V. A.; Ploug, T.; and Tao-Cheng, J.-H.: Subcellular localization of SV2 and other secretory vesicle components in PC12 cells by an efficient method of preembedding EM Immunocytochemistry for cell cultures. J. Histochem. Cytochem., 44, 1481-1488 (1996).

Du, J.; Tao-Cheng, J.-H.; Zerfas, P., and McBain, C. J.: The K+ channel, Kv2.1, is apposed to astrocytic processes and is associated with inhibitory postsynaptic membranes in hippocampal and cortical principal neurons and inhibitory interneurons. Neuroscience, 84, 37-48 (1998).

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Gold Lipids: Liposome Labels, and Precursors to Nanostructured Materials

In addition to their unique ability to selectively gold-label liposomes for light or electron microscope observation, gold cluster-labeled lipids are also potential precursors to nanostructured materials, in which the supramolecular structures formed by the lipid molecules act as templates for the organization of the attached gold particles. In our 1996 paper form the Microscopy Society of America Annual Meeting, and more fully in our 1999 paper, we describe the formation of gold-decorated liposomes:

Hainfeld, J. F.; Furuya, F. R., and Powell, R. D.: Metallosomes. J. Struct. Biol., 127, 152-160 (1999).

Abstract (Journal of Structural Biology):
See our MSA paper: www.nanoprobes.com/MSA96lip.html.

Brown and co-workers have now described the formation of Langmuir monolayers of palmitoyl Nanogold®, created by depositing an alcohol solution of nanoparticles onto deionized water at room temperature (23°C) in a Joyce-Loebl Langmuir trough. Transfer of the films to transmission electron microscope grids by the Langmuir-Schaeffer method resulted in observation of highly ordered hexagonal arrays larger than 250 x 350 nm2. Reference:

Brown, J J.; Porter, J. A.; Daghlian, C. P., and Gibson, U. J.: Ordered Arrays of Amphiphilic Gold Nanoparticles in Langmuir Monolayers. Langmuir, 17, 7966 (2001).

Journal web site: http://pubs.acs.org/journals/langd5/index.html

Highly monodisperse gold particles can form ordered arrays even without the templating action of a conjugate molecule: in our paper, from Microscopy & 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

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Very Large Gold and Metallodielectric Nanoparticles

Interested in very large gold particles (larger than 40 nm)? They can be used as carriers for nucleotides in "gene gun" experiments, for rapid diagnostics, for wavelength-specific light scattering, or for surface plasmon resonance spectroscopy. However, they are less easily stabilized than their smaller counterparts, and their size distribution is more difficult to control.

Conventionally, colloidal gold particles up to 150 nm in diameter are made by the reduction of tetrachloroauric acid with boiling sodium citrate, and size is controlled by adjusting the ratio of these reagents:

Frens, G.: Nature Physical Science, 241, 20 (1973).

Some recent papers describe improvements and alternatives to this procedure. Brown and co-workers used 2.6 nm and 12 nm colloidal gold particles as seeds for the growth of larger particles, and achieved significantly greater monodispersity for particles up to just over 100 nm in size:

Brown, K. R.; Walter, D. G.; and Natan, M. J.: Seeding of Colloidal Au Nanoparticle Solutions. 2. Improved Control of Particle Size and Shape. Chem. Mater., 12, 306-313 (2000).

Journal web site: http://pubs.acs.org/journals/cmatex/index.html

Goia and co-workers have prepared gold particles from 90 nm up to several microns in size by the reaction of tetrachloroaurate in a solution containing gum arabic with ascorbic acid, also in a solution containing gum arabic; they used pH adjustment (which is thought to control the redox potential of the system) to control particle size. Reference:

Goia, D. V., and Matijevic, E.: Tailoring the particle size of monodispersed colloidal gold. Coll. Surf. A: Physicochemical and Engineering Aspects, 146, 139-152 (1999).

Journal web site: http://www.elsevier.nl/locate/colsurfa

Graf and van Blaaderen report an alternative approach: use silica particles and coat them with gold. They prepared particles with silica cores 99 - 244 nm in radius and gold shells 33 - 88 nm thick, by depositing gold particles onto silica microparticles, then depositing metallic gold onto them. By controlling the gold thickness, they can manipulate the surface plasmon resonance frequency over a wider range than is possible with colloidal gold. Reference:

Graf, C., and van Blaaderen, A.: Metallodielectric Colloidal Core-Shell Particles for Photonic Applications. Langmuir, 18, 524-534 (2002).

Journal web site: http://pubs.acs.org/journals/langd5/index.html

Colloidal gold - technical help: www.nanoprobes.com/TechCG.html

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Other Recent Publications

Flanagan and group use 5 and 10 nm gold to simultaneously localize FLNa and Arp3, showing that they colocalize with actin on the periphery of A7 cells but show subtle differences in their organization. Cytoskeletons were prepared for electron microscopy by washing with PHEM, extraction with 0.75 % Triton X-100 in PHEM buffer containing 1 M phalloidin and 0.01 % glutaraldehyde for 2 min, and washing with PHEM followed by fixation with 1 % glutaraldehyde in PHEM containing 0.1 M phalloidin for 10 min; the cytoskeletons were treated with sodium dodecyl sulfate and unreacted aldehydes blocked before labeling with polyclonal primary antibodies and gold-labeled secondaries. Reference:

Flanagan, L. A.; Chou, J.; Falet, H.; Neujahr, R.; Hartwig, J. H., and Stossel, T. P.: Filamin A, the Arp2/3 complex, and the morphology and function of cortical actin filaments in human melanoma cells. J. Cell Biol., 155, 511-517 (2001).

Abstract (Journal of Cell Biology):

Mosgoeller, Schöfer and co-workers use 5 nm gold for in situ hybridization localization of ribosomal rDNA and rRNA in HeLa cell nucleoli subjected to intermediate hypotonic conditions to expand and disperse nucleolar components. A digoxigenin-labeled hybridization probe was detected using a primary sheep anti-digoxigenin antibody, followed by 5 nm gold-labeled secondary donkey anti-sheep antibody. Reference:

Mosgoeller, W.; Schöfer, C.; Steiner, M.; Sylvester, J. E., and Hozk, P.: Arrangement of ribosomal genes in nucleolar domains revealed by detection of Christmas tree components. Histochem. Cell Biol., 116, 495-505 (2001).

Abstract (Histochemistry and Cell Biology):

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