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N A N O P R O B E S     E - N E W S

Vol. 7, No. 4          April 14, 2006


Updated: April 14, 2006

In this Issue:

This monthly newsletter is to inform you about techniques to improve your immunogold labeling, highlight interesting articles and novel applications of metal nanoparticles, and answer your questions. We hope you enjoy it and find it useful; as always, let us know if we can improve anything.

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Nanogold® Identifies Neuron Development Promoter

The most widely reported application of Nanogold® labeling is for pre-embedding immunogold labeling. More publications describe its use for this than for any other application, and of these, a significant proportion deal with its use in neurological research, to identify and localize molecules that are significant for neurological development and operation.

Nanogold is designed with specific advantages for this type of application:

  • Nanogold conjugates are the smallest commercially available gold immunoprobes: they penetrate and reach antigens inaccessible to other probes, up to 40 microns into cells and tissue sections. Fab' conjugates are available for highest penetration.
  • Low non-specific affinity gives minimal background.
  • Extremely uniform 1.4 nm diameter gold label and close to 1 Nanogold particle per Fab' or IgG give consistent, dense labeling.
  • Highly receptive to silver enhancement; can visualize as little as 0.1 pg of target IgG on immunoblots.
  • Gold is covalently attached to antibody remote from antigen binding region, so native immunoreactivity is preserved.
  • High stability and long shelf life: conjugates show unchanged reactivity after storage for a year.

In a recent issue of Human Molecular Genetics, Zu, Tao-Cheng and group provide another informative demonstration of these advantages. They used separate confocal fluorescent immunostaining and silver-enhanced pre-embedding Nanogold labeling to study the effects of mutant proteins on neuronal development, and identify their roles in inherited neurological disorders. The hereditary spastic paraplegias (HSPs) (SPG1-29) comprise a group of inherited neurological disorders characterized by spastic lower extremity weakness due to a length-dependent, retrograde axonopathy of corticospinal motor neurons. Mutations in the gene encoding the dynamin superfamily member atlastin-1, an oligomeric GTPase highly localized to the Golgi apparatus in the adult brain, are responsible for SPG3A, a common autosomal dominant HSP. This is distinguished by frequent early onset, suggesting that developmental abnormalities may be involved in its pathogenesis. Atlastin-1 has been implicated in intracellular membrane trafficking, but although the atlastin-1 GTPase is known to be highly localized to the Golgi apparatus in the adult brain, the effect on atlastin-1 structure and activity, or neuronal function within the central nervous system, of the mutations associated with SPG3A are not known. To study these effects, the distribution of atlastin-1 in cultured neurons was compared with the effects produced by the introduction of missense atlastin-1 mutants.

For immunogold electron microscopy, rat hippocampal and cerebral cortical neuron cultures were prepared and maintained. After 34 weeks (hippocampal neurons) or 6 days (cortical neurons), the neurons were fixed with 4% paraformaldehyde, or 2% paraformaldehyde with 0.1% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) for 30 minutes. Cells were washed with 0.1 M phosphate buffer, permeabilized and blocked with 5% normal goat serum and 0.1% saponin in PBS for 1 hour, then incubated with rabbit polyclonal anti-atlastin-1 (no. 5409; 14 mg/ml) or anti-synaptophysin (DAKA; 1:250 dilution) antibodies in blocking buffer for 1 hour; in control experiments, the primary antibody was omitted. After washing with 1% normal goat serum in PBS and 2% non-fat milk in PBS, the cells were incubated with Nanogold-conjugated anti-rabbit secondary antibodies (1 : 250 dilution) in 2% non-fat dried milk in PBS for 1 hour, then washed with 2% non-fat milk in PBS and fixed with 2% glutaraldehyde in PBS for 30 minutes. After thorough washing with PBS and distilled water, cells were silver-enhanced using HQ silver and washed again with water and 0.1 M phosphate buffer. The fixed cells were then treated with 0.2% OsO4 in 0.1 M phosphate buffer for 30 minutes, mordanted en bloc with 0.25% uranyl acetate in acetate buffer (pH 5.0) overnight, washed and dehydrated with serial concentrations of ethanol, and finally infiltrated and embedded in epoxy resins.

The confocal and electron microscopic results show that in cultured neurons, atlastin-1 is highly enriched in vesicular structures within axonal growth cones and varicosities, and at axonal branch points, indicating a functional role in axonal development. Knockdown of atlastin-1 expression in these neurons using small hairpin RNAs reduces the number of neuronal processes and impairs axon formation and elongation during development. Therefore, the "long axonopathy" observed in early-onset SPG3A could result from abnormal development of axons because of loss of atlastin-1 function.

Reference:

Zhu, P. P.; Soderblom, C.; Tao-Cheng, J. H.; Stadler, J., and Blackstone, C.: SPG3A protein atlastin-1 is enriched in growth cones and promotes axon elongation during neuronal development. Hum. Mol. Genet., 15, 1343-1353 (2006).

More information:

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Osmium and Silver: How to Avoid Etching

Etching - the removal by stains such as osmium tetroxide of silver, deposited during silver enhancement of immunogold, is an ongoing problem in immunoelectron microscopy. Silver enhancement is a chemical reduction process: silver is reduced from silver (I) ions in solution and deposited onto the gold particles as metallic silver, but it is then vulnerable to oxidation. Osmium tetroxide is a powerful oxidant, and on occasions can re-oxidize the silver back into solution. Previously, we have found that this occurs principally when uranyl acetate is applied after osmication, but it has also occurred recently even in the absence of any secondary stain or oxidizing reagent.

One frustrating aspect to etching is that it is very unpredictable - the same procedure, using the same reagents, can be fine on one occasion, but strongly etched when it is repeated. Unfortunately, no clear reason has been established for this variability: it may be due to impurities in the osmium used for osmication, slight differences in the formulation of the silver enhancement reagents (particularly reagents that include a natural product as a thickening agent, such as HQ Silver), or trace amounts of other reagents from other stages in the preparation and staining. If you find this to be a problem, we recommend the following methods to prevent it from occurring:

Use a lower osmium concentration

Burry and co-workers have found that silver etching by osmium tetroxide may be greatly reduced by using 0.1% osmium tetroxide instead of 1%; this has been found to give similar levels of staining, while significantly reducing the susceptibility of the particles to etching.

Reference:

  • Burry, R.W.: Pre-embedding immunocytochemistry with silver-enhanced small gold particles. In Immunogold silver staining: Principles, methods and applications; M. A. Hayat (Ed.), CRC Press, Boca Raton, FL (1995), p. 217-230.

Use gold toning to protect silver-enhanced gold and to prevent particle loss

Gold toning is the post-treatment of silver enhanced immunogold particles with a reagent which deposits a thin layer of gold onto their surface. Gold is resistant to etching, and this protects the silver-enhanced gold particles and renders them impervious to etching. Two procedures have been described:

(1) Arai method:

  1. After silver enhancement, wash thoroughly with deionized water.
  2. 0.05% gold chloride: 10 minutes at 4°C.
  3. Wash with deionized water.
  4. 0.5% oxalic acid: 2 minutes at room temperature.
  5. 1% sodium thiosulfate (freshly made) for 1 hour.
  6. Wash thoroughly with deionized water and embed according to usual procedure.

References:

  • Arai, R.; Geffard, M., and Calas, A.: Intensification of labelings of the immunogold silver staining method by gold toning. Brain Res. Bull., 28, 343-345 (1992).

  • Arai, R., and Nagatsu, I.: Application of Gold Toning to Immunogold-Silver Staining. In Immunogold-Silver Staining: principles, Methods and Applications; M. A. Hayat (Ed.), CRC Press, Boca Raton, FL (1995) ch. 13, pp 209-216.

(2) An alternative procedure has been reported by Sawada and Esaki:

  1. Rinse twice quickly in distilled water.
  2. 0.05 M sodium acetate (1 minute) then rinse again quickly.
  3. 0.05 % tetrachloroauric acid (2 minutes).
  4. Thorough rinsing in distilled water for 10 minutes, then osmicate.

References:

A possible disadvantage of using gold toning is that it can generate more background than silver enhancement alone. However, Suikkanen and group report a handy pre-embedding protocol using Nanogold® immunolabeling and silver enhancement with gold toning, which confers excellent thermal stability as well as resistance to osmium etching. This was used to study the effects of endocytosis-modulating drugs upon the release of canine parvovirus (CPV) from endosomal vesicles during infection of cultured cells, and the role of phospholipase A2 (PLA2) in this process; electron microscopic localization of the virus after exposure to the different drugs showed that PLA2 activity is essential, and suggested that while endosomes remain intact after infection, it causes changes in permeability which are essential for release.

Norden Laboratories feline kidney cells were incubated with CPV (m.o.i. 50) in Dulbeccos modified Eagles medium for 20 h in the presence or absence of the drugs; when used, they were added on the top of the cells 30 min before virus and maintained until fixation.

The pre-embedding procedure is:

  1. After 120 hours, wash dishes with 0.1 M phosphate buffer, pH 7.4. Fix cells with prednisolone phosphate (PLP) for 2 hours at room temperature.
  2. After rinsing, permeabilize cells for 8 min at room temperature with phosphate buffer containing 0.01% saponin and 0.1% bovine serum albumin, or with 0.05% Triton X-100 in 0.1 M phosphate (experiments to visualize nuclear antigens).
  3. Incubate primary antibody (MaCPV or RaVP1) on the top of the cells for 1 hour at room temperature.
  4. Wash with permeabilization buffer.
  5. Incubate with Nanogold-labeled IgG (anti-rabbit or anti-mouse) secondary antibody on the top of the cells for 1 hour at room temperature.
  6. Wash with permeabilization buffer.
  7. Postfix with 1% glutaraldehyde in 0.1 M phosphate buffer for 10 minutes at room temperature. Quench with 50 mM ammonium chloride in phosphate buffer, and wash with both phosphate buffer and water.
  8. Enhance in the dark with HQ Silver for 2 minutes, then wash thoroughly with water.
  9. 2% sodium acetate, 3 X 5 minutes.
  10. 0.05% gold chloride 10 minutes on ice.
  11. 0.3% sodium thiosulfate 2 X 10 minutes on ice.
  12. Wash with water.
  13. Postfix with 1% osmium tetroxide in 0.1 M phosphate buffer for 1 hour at 4°C.
  14. Dehydrate with a descending concentration series of ethanol.
  15. Stain with 2% uranyl acetate.
  16. Embed by placing plastic capsules filled with Epon LX-112 upside-down on top of the cells. After polymerization, warm up to 100°C, carefully remove capsules, and cut 50 nm horizontal sections.
  17. Stain with 2% uranyl acetate and lead citrate.

Reference:

  • Suikkanen, S.; Antila, M.; Jaatinen, A.; Vihinen-Ranta, M., and Vuento, M.: Release of canine parvovirus from endocytic vesicles. Virology, 316, 267-280 (2003).

Use Gold enhancement instead of silver enhancement

Gold Enhancement is a novel autometallographic process, developed at Nanoprobes, in which gold rather than silver is deposited. Gold is not susceptible to etching by osmium, and therefore using gold enhancement in place of silver will ensure that the enhanced particles resist even strongly oxidizing poststaining conditions.

There are other reasons to consider GoldEnhance. In addition to osmium etch resistance, it has a number of other advantages:

  • GoldEnhance may be used in physiological buffers (chlorides precipitate silver, but not gold).
  • The gold autometallographic reaction is less pH sensitive than that of silver.
  • Gold gives a much stronger backscatter signal than silver for SEM work.
  • GoldEnhance has near neutral pH, unlike some silver enhancement reagents that are quite acidic, and has a relatively low ionic strength. These factors support high ultrastructural preservation.
  • It has low viscosity, so the components may be dispensed and mixed easily and accurately.
  • Gold enhancement can be used in environments where silver would be precipitated, such as enhancement of gold labeling in cells cultured on metal substrates.

More information:

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Nanogold® with LI Silver for CryoEM and Gel Staining

In addition to its electron microscopic applications, Nanogold® with silver enhancement provides a highly sensitive and specific detection and visualization method for blots, gels, and light microscopy. We have discussed the application of Nanogold to gel staining in a previous article, and our web site include an application note which describes the differences between Nanogold with silver enhancement and other gel staining methods.

Yonekura and group presented a paper in the Journal of Molecular Biology which has both: high-resolution cryo-electron microscopy using Nanogold labeling, and gel staining using LI Silver to visualize Nanogold-labeled proteins. A motor protein complex, PomA/B, from the bacterial flagellum, Vibrio alginolyticus, was reconstituted into liposomes and visualized by electron cryomicroscopy. PomA/B is a sodium channel, composed of two membrane proteins, PomA and PomB. It converts ion flux to the rotation of the flagellar motor. Escherichia coli and Salmonella have a homolog, MotA/B, which utilizes protons instead of sodium ions. PomB and MotB are regarded as the stator since they both have a peptidoglycan-binding motif in their C-terminal region. Energy filtering electron cryomicroscopy was used to enhance the contrast between the structures of different elemental composition; this enhanced the image contrast of the proteins reconstituted into liposomes, and showed that two extramembrane domains with clearly different sizes stick out of the lipid bilayers on opposite sides.

To confirm that the particles were the PomA/B complex, Monomaleimido Nanogold was used. PomB has three free cysteine residues, two in the N-terminal region (Cys10 and Cys8) and one in the transmembrane region (Cys31), whereas PomA has no cysteine. For gold labeling of cysteine, Monomaleimido Nanogold was added to the proteoliposomes at a final concentration of Nanogold at a few times excess to the protein, and the samples were incubated at room temperature for 1 hour. After dialysis to remove unbound Nanogold at 4°C for a few days, the samples were run on 12.5% or 15% (w/v) homogeneous polyacrylamide gels using the Laemmli buffer system: To check that Monomaleimido Nanogold was bound to the cysteine residues, the buffer system contained essentially no reductant. The gels were developed with an LI Silver stain kit (Nanoprobes, Yaphank, NY) to detect bound Nanogold. Labeling was done in the absence of reducing agent; under this condition, PomB forms a dimer with a disulfide bond, but this does not affect the motor function. LI silver was then used to visualize the bound Nanogold: the band corresponding to the PomB dimer was clearly stained. Regions corresponding to the smeared band of the PomA monomer and the region below are also stained but much more weakly, possibly due to non-specifically bound and unbound gold respectively.

In the cryo-EM image of the gold-labeled protein, individual gold particles were visible at relatively small defocus, e.g. atw7000 Å, but were much harder to see at larger defocus, such as a few microns. In contrast, proteins on the surface of liposomes were almost impossible to visualize at small defocus even by energy filtering. One solution was to record two identical views at different defocus levels; however, in practice the second shot frequently destroyed the lipid vesicles even at relatively low dose. However, averaging of about ten images clearly revealed the bound gold particle. The density distribution of Nanogold was broad due to the rough alignment of heterogeneous images, but the high-density region is clearly located on the surface of the membrane on the shorter domain side. This means that Cys31 was not labeled since it is predicted to be located near the center of the membrane rather than on the membrane surface. Cys31 is not responsible for dimer formation: therefore, either Cys8 or Cys10 must be forming a disulfide bond, while the other is labeled with Nanogold. This indicates that the side of the shorter domain is most likely the PomB N-terminal side.

[Nanogold labeling reagents, labeling of PomA/B, and blot (77k)]

(a): Monomaleimido Nanogold® and Mono-Sulfo-NHS-Nanogold labeling reagents and conjugation reactions. (b): Monomaleimido Nanogold labels an external cysteine residue in liposomal Pom A/B complex. (b): Immunodot blot showing the detection of serial dilutions of mouse IgG on a nitrocellulose membrane, using Nanogold-anti-mouse Fab' developed with LI Silver. The Nanogold particles nucleate silver deposition so well that unprecedented sensitivity is achieved: the last spot detected contains just 0.1 pg of IgG target (arrow).

Deletion of peptidoglycan-binding motif produced a particle without the large periplasmic domain. Combining the image analysis with gold labeling, with the results after deletion of the peptidoglycan-binding motif, revealed that the longer one, ~70Å long, is likely to correspond to the periplasmic domain, and remaining structure, about half size, to the cytoplasmic domain.

Reference:

Yonekura, K.; Yakushi, T.; Atsumi, T.; Maki-Yonekura, S.; Homma, M., Namba, K.: Electron cryomicroscopic visualization of PomA/B stator units of the sodium-driven flagellar motor in liposomes. J. Mol. Biol., 357, 73-81 (2006).

One important note on gel staining: the silver enhancement reagents and staining procedure are not the same as the "silver staining" procedure used for general visualization of proteins on gels. With silver staining, the proteins are stained directly by treatment with formaldehyde and a silver salt; it is a different chemical process to silver enhancement, which selectively visualizes gold labels, and does not stain proteins in the absence of the gold. In the formaldehyde silver staining system, the silver is deposited directly onto the biological molecule in question, and all protein molecules on the gel are stained and visualized. Nanoprobes does not manufacture silver staining reagents of this type. For details on silver staining and its mechanism, see:

Rabilloud, T.: Mechanisms of Protein Silver Staining in Polyacrylamide gels: A 10-year Synthesis. Electrophoresis, 11, 785-794 (1990).

More information:

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Nanogold® Helps Elucidate Endocytic and Secretory Trafficking in Arabidopsis

In eukaryotic cells, compartments of the dynamic endomembrane system are acidified to varying degrees by the activity of vacuolar H-ATPases (V-ATPases). In Arabidopsis thaliana, most V-ATPase subunits are encoded by small gene families; this means that many enzyme complexes are possible, with different kinetic properties and localizations. York-Dieter Stierhof and colleagues describe, in a recent issue of The Plant Cell, their determination of the subcellular localization of the three Arabidopsis isoforms of the membrane-integral V-ATPase subunit VHA-a, using Nanogold-Fab' conjugates to localize them at the ultrastructural level; they also report a novel approach to combined fluorescent and gold labeling, i.e. the silver enhancement of quantum dots.

Immunogold labeling was performed on ultrathin thawed Tokuyasu cryosections of formaldehyde-fixed (8%, 3 hours) and sucrose-infiltrated (2.1 M) root tips using rabbit anti-GFP serum (1 : 25, Abcam) or rabbit anti-VHAE serum (1 : 500), followed by Nanogold®-labeled Fab' goat anti-rabbit IgG, and enhancement for 6 minutes using HQ Silver. In parallel, GFP-labeled NST-GFPexpressing root cells were labeled with a Quantum dot-conjugated goat F(ab')2 anti-rabbit IgG (coupled to Qdot525; 1 : 20), which was then silver-enhanced using HQ Silver (5 minutes). For structural analysis, root tips were either chemically fixed with 2.5% glutaraldehyde, 1% aqueous uranyl acetate, and 1% osmium tetroxide, dehydrated, and embedded in Epon, or high-pressure frozen in hexadecane, freeze-substituted (72 hours at -90°C; 8 hours at -60°C; 8 hours at -35°C; 4 hours at 0°C) in acetone containing 2% osmium tetroxide and 0.5% uranyl acetate, washed at 0°C, and embedded in Epon.

Fluorescence colocalization using quantum dots and GFP expression, as well as immunogold labeling, showed that VHA-a1 is preferentially found in the trans-Golgi network (TGN), which is the main sorting compartment of the secretory pathway. Uptake experiments with the endocytic tracer FM4-64 revealed rapid colocalization with VHA-a1, indicating that the TGN may act as an early endosomal compartment. Concanamycin A, a specific V-ATPase inhibitor, blocked endocytic transport of FM4-64 to the tonoplast, causes the accumulation of FM4-64 together with newly synthesized plasma membrane proteins, and interferes with the formation of brefeldin A compartments. Nascent cell plates are also rapidly stained by FM4-64, indicating that endocytosed material is redirected into the secretory flow after reaching the TGN. Taken together, these results suggest the convergence of the early endocytic and secretory trafficking pathways in the TGN.

Reference:

Dettmer, J.; Hong-Hermesdorf, A.; Stierhof, Y.-D., and Schumacher, K.: Vacuolar H+-ATPase activity is required for endocytic and secretory trafficking in Arabidopsis. Plant Cell, 18, 715-730 (2006).

More information:

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Nanoprobes Address Change

Because of a reorganization in the building where we are located, we have added "unit 1" to our address. Please use this to make sure that your mail gets to us:

Nanoprobes, Incorporated
95 Horseblock Road, Unit 1
Yaphank, NY 11980-2301
USA

We have also upgraded our telephone system; you may have noticed our new call routing. The extensions for several of our offices and staff have been changed during this process, so if you want to talk to a specific person directly, you may need to dial a different extension number. Our people page has updated extensions and contact information, or you can access an updated directory listing when you call us.

Our principal contact numbers remain the same, and for your information, contact information is summarized below:

Question: Contact Telephone E-mail
Ordering, order status, shipping or payment Sales office 1-877-447-6266 or (631) 205-9490 nano@nanoprobes.com
Product availability or delivery time Sales office 1-877-447-6266 or (631) 205-9490 nano@nanoprobes.com
Technical or application question Technical support 1-877-447-6266 or (631) 205-9492 tech@nanoprobes.com
Problem with product Technical support 1-877-447-6266 or (631) 205-9492 tech@nanoprobes.com
Business inquiry or general information General business office 1-877-447-6266 or (631) 205-9490 nano@nanoprobes.com

More information:

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Other Recent Publications

Hong and Kand describe the use of metal nanoparticles for enhance fluorescence in their recent report in Biosensors and Bioelectronics. Fluorophores have been used as effective signal mediators for detecting biomarkers; the authors used enhancement of the fluorescence by 2, 5 and 10 nm colloidal gold particles coated with a self-assembled monolayer of L-glutathione (1 nm thickness) or 16-mercaptohexadecanoic acid (2 nm thickness) to improve the sensitivity of fluorophore-mediated biosensors. Gold particles, when placed at an appropriate distance from a fluorophore, can effectively enhance the fluorescence by transferring the free electrons of the fluorophore, normally used for self-quenching, to the strong surface plasmon polariton field (SPPF) of the gold particle. They found that some organic solvents, specifically ethanol, can also enhance the fluorescence significantly, while the degree of enhancement was largest with the smaller sizes. To maximize the fluorescence enhancement, novel, biocompatible nanogold particle reagents (NGPRs) were prepared by combining NGPs and biocompatible solvents, then tested. The level of enhancement by NGPRs was found to be additively contributed by two enhancers. These reagents were able to increase the signal of a fiber-optic biosensor as much as 10 times, and accurately quantify cardiac markers at a tens of picomolar level; these novel enhancers may be effective for fluorophore-mediated bioimaging as well as biosensing. We do hope, however, that they would refrain from describing them as "Nanogold" - this is our trademark.

Reference:

Hong, B., and Kang, K. A.: Biocompatible, nanogold-particle fluorescence enhancer for fluorophore mediated, optical immunosensor. Biosens. Bioelectron., 21, 1333-1338 (2006).

Brust and group report the preparation of stable suspensions of size-uniform 40 - 50 nm spherical assemblies of 5-8 nm gold colloids in toluene by cross linking the colloidal particles using alkanedithiols, within a defined range of gold-dithiol molar ratios. The assemblies are very stable and remain suspended in toluene for several months without significant aggregation; they were characterized by transmission electron microscopy, UV/visible spectroscopy, and atomic force microscopy. These porous gold spheres can be further organized into hierarchically assembled relatively linear chains by the addition of ethanol.

Reference:

Hussain, I.; Wang, Z.; Cooper, A. I., and Brust, M.: Formation of Spherical Nanostructures by the Controlled Aggregation of Gold Colloids. Langmuir, 22, 2938-2941 (2006).

Electrical detection is also a hot topic these days, and Hansen and co-workers add to it with their recent report that metal sulfide nanoparticles can provide femtomolar levels of detection when used as electrochemical sensors. Cadmium, zinc and lead sulfide nanoparticles were conjugated to 5'-thiolated reporter oligonucleotides. These were then bound using immobilized complementary "capture" oligonucleotides. The metal sulfide particles were then dissolved, and the metal content of the resulting solution determined using anodic stripping voltammetry, a highly sensitive electrochemical detection method. Decreasing the amount of target DNA from 50 to 0.1 fmol in a series of experiments revealed the very high sensitivity of the method: with one target, the sensor proved capable of efficiently detecting down to 0.1 fmol (33 fM, 3 mL) of the target.

Reference:

Hansen, J. A.; Mukhopadhyay, R.; Hansen, J. O.; and Gothelf, K. V.: Femtomolar electrochemical detection of DNA targets using metal sulfide nanoparticles. J. Amer. Chem. Soc., 128, 3860-3861 (2006).

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