Updated: May 13, 2003

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

Vol. 4, No. 5 May 13, 2003

This monthly newsletter is to 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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Gold Cluster Labels in Cryo-EM: Structure of Filamentous Phage 4

Nanogold® has been extensively used for cryo-EM, where its twin advantages of high precision labeling and high resolution staining enable realization of the full accuracy of the method. Covalent linking to specific sites at the molecular level gives high precision; because the gold label does not require additional macromolecules for stabilization, conjugates may be prepared much smaller than conventional colloidal gold probes, allowing more dense labeling, increased antigen access, and closer positioning of the label to the target.

Opalka and co-workers demonstrate these advantages in their determination of the structure of filamentous Phage IV (pIV) by cryo-EM associated with image analysis. The structure reveals a barrel-like complex, 13.5 nm in diameter and 24 nm in length, with D14 point-group symmetry, consisting of a dimer of unit multimers. Side views of each unit multimer exhibit three cylindrical domains named the N-ring, the M-ring and the C-ring. pIV was engineered to contain a single cysteine residue near the N or the C terminus; these were labeled using monomaleimido Nanogold while dissolved in 1% (w/v) CHAPS, 20 mM NaPO₄ (pH6.5), 500 mM NaCl with 1 mM EDTA, at 4°C overnight. Unconjugated Nanogold was separated using 3-4 buffer washes on a Centricon-100 membrane centrifuge filter. Cryo-EM of labeled pIV unambiguously identified the N-terminal region as the N-ring, and the C-terminal region was inferred to make up the C-ring.

Reference:

Opalka, N.; Beckmann, R.; Boisset, N.; Simon, M. N.; Russel, M., and Darst, S. A.: Structure of the filamentous phage pIV multimer by cryo-electron microscopy. J. Mol. Biol., 325, 461-470 (2003).

Abstract (courtesy of Elsevier/Science Direct):
http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6WK7-47G321T-7&_coverDate=01%2F17%2F2003&_alid=91572617&_rdoc=1&_fmt=&_orig=search&_qd=1&_cdi=6899&_sort=d&view=c&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=1facc2003e3a13245b0624823009729c

Image analysis increases the resolution of structural information available from the sample, and this in turn means that smaller metal cluster labels with even higher resolution may be localized. Undecagold, and even the yet smaller tetrairidium cluster, have been used to achieve even higher resolution labeling, and these smaller labels also result in even less perturbation of the native structure.

Reference:

Cheng, N.; Conway, J. F.; Watts, N. R.; Hainfeld, J. F.; Joshi, V.; Powell, R. D.; Stahl, S. J.; Wingfield, P. E., and Steven, A. C. Tetrairidium, a 4-atom Cluster, is Readily Visible as a Density Label in 3D Cryo-EM Maps of Proteins at 10 - 25 Å Resolution. J. Struct. Biol., 127, 169-176 (1999).

Abstract (courtesy of the Journal of Structural Biology):
http://www.idealibrary.com/links/artid/jsbi.1999.4120/production

More information:

Nanogold labeling - catalog information: http://www.nanoprobes.com/LabRgts.html
Other Nanogold applications: http://www.nanoprobes.com/Applic.html
Product info - Mono-Sulfo-NHS-Nanogold: http://www.nanoprobes.com/Inf2025.html
References - Nanogold cryo-EM: http://www.nanoprobes.com/RefTopNG.html#Ncryosec
References - Undecagold cryo-EM: http://www.nanoprobes.com/RefTopUG.html#Ucryo
Technical help - Nanogold labeling: http://www.nanoprobes.com/TechNGlr.html

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Finding Solutions to some Immunogold Labeling Problems

(1) Background or non-specific gold binding

Background, or 'non-specific' staining occurs when the gold conjugate binds to other components of your specimen as well as, or instead of, the intended target. If it cannot be removed by adjusting the concentrations of the staining reagents, the best approach is to identify where it is occurring, infer what the mechanism might be, and use this to find a blocking or washing method.

Generally, we find that 5% nonfat dried milk is effective for reducing general background. This may be used as a blocking solution before incubation, but is even more effective if it is included in the incubation buffer with the Nanogold conjugate.
Does the distribution of the binding suggest a blocking method? For example, if it occurs in thiol (cysteine)-rich regions, it may be due to thiol coordination to the gold. This may be blocked using N-ethylmaleimide. If it occurs in nuclear material, it may be due to interactions with the ionic charges of nucleic acids; increasing the ionic strength of the buffer, or changing the pH to a value at which the nucleic acids are less ionized, may help.
Could something from the fixation cause binding? if residual aldehydes are present, them may react with the antibody. These should be thoroughly blocked with glycine, ammonium chloride, or sodium cyanoborohydride before immunostaining.
If there is no obvious pattern to the background, a wash solution that solubilizes gold can help make sure all the gold is removed. Background binding is often attributed to hydrophobic interactions, and therefore adding reagents that reduce hydrophobic interactions to the wash buffer may help remove it. Examples include 0.6 M triethylammonium bicarbonate buffer (prepared by bubbling carbon dioxide into an aqueous suspension of triethylamine with stirring: 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; and 0.1 % to 0.5 % of an amphiphile, such as benzamidine or 1,2,3-trihydroxyheptane. 20 % DMSO in aqueous buffer is also effective for solubilizing Nanogold.

(2) Low labeling

Due to its small size and because it does not require additional macromolecules for stabilization, Nanogold conjugates are very small, and can often label targets that are inaccessible to conventional colloidal gold probes. If you find low labeling with a Nanogold conjugate, the following suggestions may be helpful:

It is usually worth checking by light microscopy first, using silver or gold enhancement to visualize the gold. If immunogold staining does not work, do the same conditions yield effective staining with a fluorescent or enzymatic probe? If they do, there may be a problem with the Nanogold probe. Test in a blot: spot serial dilutions of target primary antibody (first spot 0.1 micrograms) onto a hydrated nitrocellulose membrane, incubate with a 1 : 50 to 1 : 200 dilution of the Nanogold conjugate, wash, and develop with silver or gold enhancement. If no signal develops on the blot, the Nanogold conjugate is not binding to its target.
If the blot develops successfully, it is helpful to remember that immunolabeling requires a compromise between better preservation of the ultrastructure (better fixation) and easier antigen access (more permeabilization and less fixation). Generally, it is better to begin with a relatively low level of fixation so labeling works, then increase fixation conditions to find the best preservation at which you still obtain good labeling.

More information:

Technical help - Nanogold conjugates: http://www.nanoprobes.com/TechNGAb.html
References - Nanogold labeling: http://www.nanoprobes.com/RefTopNG.html
Technical Help - ImmunoEM: http://www.nanoprobes.com/TechIEM.html

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Nanogold® Labeling of Bak: Role in Mitochondrial Permeability

Mikhailov and co-workers have used Nanogold®-Fab' to localize Bax and elucidate its role in ATP-deprived rat proximal tubule cells. For pre-embedding EM immuno-cytochemistry in normal and apoptotic cells after ATP depletion and repletion, cells were fixed with 4% formaldehyde in 0.1 M phosphate buffer at pH 7.4 for 45 min, washed, saponin-permeabilized, and incubated with anti-Bax antibody (1D1) followed by Nanogold-labeled Fab' secondary antibody. Silver enhancement (HQ Silver) was applied, then samples were treated with 0.2% OsO₄, washed, stained with uranyl acetate, dehydrated in ethanol, and embedded in Epoxy resin.

The authors found that Bax and the closely associated Bak both form homo-oligomers in ATP-depleted cells, and showed that Bax is required for Bak homo-oligomerization and for cytochrome c release during energy deprivation. This implies a degree of functional cooperation between Bax and Bak in this form of cell injury; the results also show that Bax is required for mitochondrial permeabilization.

Reference:

Mikhailov, V.; Mikhailova, M.; Degenhardt, K.; Venkatachalam, M. A.; White, E., and Saikumar, P.: Association of Bax and Bak homo-oligomers in mitochondria. Bax requirement for Bak reorganization and cytochrome c release. J. Biol. Chem., 278, 5367-5376 (2003).

Abstract (courtesy of the Journal of Biological Chemistry):
http://www.jbc.org/cgi/content/abstract/278/7/5367

More information:

Catalog information - Nanogold conjugates: http://www.nanoprobes.com/NanoAb.html
References - Nanogold labeling: http://www.nanoprobes.com/RefTopNG.html
Product info - Nanogold-antibodies: http://www.nanoprobes.com/Inf2001.html

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Double Labeling: Combining Silver-Enhanced Gold with other Labels

We are occasionally asked whether you can combine gold labeling with silver or gold enhancement with another labeling or staining method. Double labeling using silver-enhanced gold to mark one site of interest and chromogenic enzymatic staining to mark a second has been described for electron microscopy; the two staining patterns are readily identifiable in the microscope.

If you decide to try this procedure, you should complete the Nanogold labeling and silver or gold enhancement, then washing thoroughly before the peroxidase/DAB labeling. This will prevent the reagents used with DAB/peroxidase staining nucleating silver or gold deposition during enhancement and generating non-specific signal.

References:

Bernard, V.; Levey, A. I., and Bloch, B.: Regulation of the subcellular distribution of m4 muscarinic acetylcholine receptors in striatal neurons in vivo by the cholinergic environment: evidence for regulation of cell surface receptors by endogenous and exogenous stimulation. J. Neurosci., 19, 10237-10249 (1999).
Abstract (courtesy of the Journal of Neuroscience):
http://www.jneurosci.org/cgi/content/abstract/19/23/10237
Li, H.; Ohishi, H.; Kinoshita, A.; Shigemoto, R.; Nomura, S., and Mizuno, N.: Localization of a metabotropic glutamate receptor, mGluR7, in axon terminals of presumed nociceptive, primary afferent fibers in the superficial layers of the spinal dorsal horn: an electron microscope study in the rat. Neuroscience Lett., 223, 153-156 (1997).
Abstract (Medline):
http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=9080455&dopt=Abstract
Salas, P. J. I.: Insoluble gamma-tubulin-containing structures are anchored to the apical network of intermediate filaments in polarized CACO-2 epithelial cells. J. Cell Biol., 146, 645-657 (1999).
Abstract (courtesy of the Journal of Cell Biology):
http://www.jcb.org/cgi/content/abstract/146/3/645

Visit the references section of our web site to see some of the other applications of silver and gold enhancement. Like all our reference pages, the one for silver and gold enhancement is available sorted by product or by application:

More information:

References by application: http://www.nanoprobes.com/RefTopSE.html
References by product: http://www.nanoprobes.com/Refsilver.html
Tech Help - silver enhancement: http://www.nanoprobes.com/TechSE.html

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Other Recent Publications

Ren and co-workers describe a technique with a high success rate for correlative imaging of the same cell using light or fluorescence microscopy, and either standard or energy-filtered electron microscopy, using 50-mesh grids and Quetol (low autofluorescence) resin. This is particularly appropriate for high-resolution, high-sensitivity localization of different metal nanoparticle labels.

Reference:

Ren, Y.; Kruhlaka, M. J., and BazettJones, D. P.: Same Serial Section Correlative Light and Energy-filtered Transmission Electron Microscopy. J. Histochem. Cytochem., 51, 605-612 (2003).

Abstract (courtesy of the Journal of Histochemistry and Cytochemistry):
http://www.jhc.org/cgi/content/abstract/51/5/605

Mayhew and group describe a simpler method for estimating immunogold labeling densities (without estimating areas or lengths). The observed and expected LD values can then be used to calculate a relative labelling index (RLI) for each compartment and then test statistically for preferential (non-random) labelling.

Reference:

Mayhew, T.; Griffiths, G.; Habermann, A.; Lucocq, J.; Emre, N., and Webster, P.: A simpler way of comparing the labelling densities of cellular compartments illustrated using data from VPARP and LAMP-1 immunogold labelling experiments. Histochem. Cell Biol., 119, 333-341 (2003).

Abstract (courtesy of Springer):
http://link.springer.de/link/service/journals/00418/contents/03/00523/

The patterning of metal nanoparticles is an area of considerable current interest, and Tanaka and co-workers find that that p(DDA/VPy) LB films act as a good template for gold nanoparticle arrays. Ultrathin films of poly(N-dodecyl acrylamide-co-4-vinyl pyridine)s (p(DDA/VPy)s) copolymers were prepared by the Langmuir-Blodgett (LB) technique. Gold nanoparticles were immobilized onto p(DDA/VPy) LB films through the dipping method and took a uniformly distributed monolayer formation; the amount of gold nanoparticle in the monolayer strongly depended on the VPy content. A patterned gold nanoparticle monolayer was obtained with photopatterned p(DDA/VPy) LB films.

Reference:

Tanaka, H.; Mitsuishi, M.,and Miyashita, T.: Tailored-Control of Gold Nanoparticle Adsorption onto Polymer Nanosheets. Langmuir, 19, 3103-3105 (2003).

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