N A N O P R O B E S E - N E W S
Vol. 5, No. 11 November 5, 2004
In this Issue:
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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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We are frequently asked what the concentration of our Nanogold® conjugates is. The short answer is that it is 80 micrograms per mL of protein: this does not include the mass of the attached gold particle. This translates to about 0.6 nmol (nanomoles) per mL of IgG conjugates, 1.6 nmol/mL of Fab', or about 1.3 nmol/mL of streptavidin.
It's worth noting how we calculate this, because you can use the same method for calculating the concentration of conjugates that you prepare using our Nanogold labeling reagents: spectroscopically, using the UV/visible absorption spectrum of the conjugate and the known extinction coefficients of the Nanogold particle and the antibody. The basis for the calculation is that we subtract the absorption due to the gold from that of the conjugate to find that of the conjugate protein at its peak absorption wavelength (usually 280 nm), and, using the extinction coefficient of the protein, use this to calculate its concentration. This is then used to dilute the conjugate to exactly 0.08 mg/mL conjugate protein concentration for commercial packaging.
![[Spectra of Nanogold and conjugate] (5k)]](../Images/NConjsp2.gif)
UV/visible absorption spectrum of Nanogold (black) overlaid with spectrum of a Nanogold-Fab' conjugate (red - difference region), scaled to the same absorption in the Nanogold-only regions. Subtraction of the Nanogold-only absorption at 280 nm from that of the Nanogold-Fab' conjugate gives the absorption of the Fab' only, which is then used to calculate the amount of Fab' present and determine the dilution for packaging.
A step-by-step explanation of this calculation is provided on our web site, which you can use to calculate both the concentration and labeling of our own conjugates: this uses the absorption and extinction coefficient of Nanogold at 420 nm, where most proteins do not absorb, to calculate the Nanogold concentration. If your protein does absorb in this region, we also provide a more rigorous method for calculating the concentration and degree of labeling of your product. In addition, a previous article gives full step-by-step instructions for the first method.
For peptides and other small molecules whose absorption is very small relative to the Nanogold, concentration is very difficult to measure because small variations or even noisiness in the conjugate spectrum greatly affects the results. In these cases, the concentration of Nanogold is a much more reliable indicator of conjugate concentration.
More information:
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Negative Staining: NanoVan in Electron Microscopy of Microsporida
Unlike 'positive' stains, which stain regions of specimens, negative stains fill in the gaps between features and contrast the edges of particulate or suspended specimens such as protein complexes or cells. Nanoprobes offers two negative stain reagents with complementary properties. NanoVan is recommended for use with Nanogold® because it is based on vanadium and is therefore less electron-dense than heavy metal based stains such as uranyl acetate or lead citrate. Nano-W is based on the heavier element tungsten, and therefore gives a more dense stain, and is more suited to use with larger gold labels.
Advantages of these reagents:
- Completely miscible: they may be mixed in different proportions to give desired intermediate stain density.
- Near-neutral pH results in better ultrastructural preservation.
- NanoVan is less susceptible to electron beam damage than uranyl acetate.
- Fine grain allows high imaging resolution.
While negative stains are frequently applied to isolated protein complexes, they can also be highly effective when used with gold labeling to pinpoint surface features of cellular components. Xu, Takvorian and group used NanoVan to study the role of glycosylation of the polar tube of Microsporida, a class of eukaryotic spore-forming parasites, in the infection of their host. The polar tube, through which the sporoplasm is transferred to the host cell, is the defining structure of microsporidia. Three polar tube proteins, PTP1, PTP2, and PTP3, are identified. The majority protein, PTP1, was found to be post-translationally modified, and Concanavalin A (ConA) was found to bind to both PTP1 and to the polar tube of several different microsporidia species. Analysis of N- and O- linked glycosylation of Encephalitozoon hellem PTP1 using deglycosylation kits showed that it is modified by O-linked mannosylation. ConA binds to O-linked mannose residues, and this enabled the electron microscopic localization of glycosylation sites in the germinating microsporida using gold-labeled ConA.
While mannose pretreatment of RK13 host cells decreased their infection by E. hellem, consistent with an interaction between the mannosylation of PTP1 and some unknown host cell mannose binding molecule, a CHO cell line (Lec1) that is unable to synthesize complex-type N-linked oligosaccharides had an increased susceptibility to infection compared to wild-type CHO cells. This suggests that PT1 O-mannosylation has functional significance for the ability of microsporidia to invade host cells.
For lectin staining, 10-microliter aliquots of each spore type were placed in two microcentrifuge tubes and twenty microliters of germination buffer (140 mM NaCl, 5 mM KCl, and 1 mM CaCl2 in 0.05 M Tris-HCl buffer, pH 8.6) added to each tube (one experimental, one control) containing B. algerae spores. 20 microliters of 3% hydrogen peroxide was added to the two tubes (experimental and control) of E. hellem spores. After 3 min, 20 microliters of Concanavalin A (ConA) labeled with 15-nm colloidal gold, diluted 1:20 in PBS containing 0.002 M magnesium chloride and 0.002 M calcium chloride, was added to the respective experimental tubes. In the control tubes, 10 microliters of 0.2 M alpha-mannopyranoside (EY Laboratories) was added to the germinating spores just prior to addition of the ConA. The germinated spores were allowed to react with the ConA for 30 min at room temperature. At the end of the incubation period, 40 microliters of PBS was added to each tube and the contents were gently mixed by inversion. The tubes were then centrifuged until a soft pellet formed, and the supernatant carefully removed. Centrifugation and supernatant removal was carried out five times, using PBS as a wash: washes used for controls contained 0.2 M alpha-mannopyranoside. Each of the four pellets was resuspended in 40 microliters of PBS. 3 microliters of the respective suspension was pipetted onto formvar-coated grids and allowed to settle for 1 min. After settling, an equal amount (3 microliters) of NanoVan was added to each grid, and after 5 min the liquid was carefully wicked away with filter paper. The grids were air-dried overnight and the samples were then observed and photographed with a Phillips Tecnai 12 operated at 80 kV at.
Reference:
Xu, Y.; Takvorian, P. M.; Cali, A.; Orr, G., and Weiss, L. M.: Glycosylation of the major polar tube protein of Encephalitozoon hellem, a microsporidian parasite that infects humans. Infect. Immun., 72, 6341-6350 (2004).
More information:
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Receptor Recycling Elucidated with Nanogold® Double Labeling
Nanogold®-Fab' is the smallest immunogold conjugate routinely available, and therefore has the highest sample penetration, labeling density and ability to access and label otherwise inaccessible antigens. Pagano and co-workers used all these features to their advantage in studies of receptor recycling in cultured cells, and also described a novel double labeling method in which one target stained with silver-enhanced Nanogold was distinguished from a second marked with 10 nm colloidal gold by its lower density.
The object of the current study was to determine whether clathrin and associated adaptor proteins are involved in receptor recycling from endosomes back to the plasma membrane, using an in vitro assay to identify the molecular requirements for the formation of recycling vesicles. MDCK (strain II) cells stably expressing the human asialoglycoprotein (ASGP) receptor subunit H1, a typical recycling receptor, with a C-terminal myc-tag were surface biotinylated and then allowed to endocytose for 10 min. Surface biotin was then removed, the cells permeabilized, and the cytosol washed away; the formation of sealed vesicles containing biotinylated H1 could then be reconstituted in a temperature-, cytosol-, and nucleotide-dependent manner.
Cryoelectron microscopy was used to test for the presence of two targets in vesicles: H1 internalized from the plasma membrane to endosomes before permeabilization of the cells, which was detected using silver-enhanced Nanogold-F(ab') fragments, and AP-1 detected with anti-gamma-adaptin antibody and 10 nm colloidal gold - protein A. Labeled membranes generally had a vesicular appearance with a diameter of ~100 nm. Coat structures were not obvious on any of the membranes in the sample, but vesicles were found that were positive for both colloidal gold and Nanogold; this is consistent with the biochemical evidence that H1 is released from endosomes in vesicles involving AP-1containing coats.
The Nanogold conjugate was prepared using Monomaleimido Nanogold to label F(ab') fragments of anti-ASGP receptor antibodies. The IgG fraction of a polyclonal antiserum was isolated with protein A-Sepharose, digested with 4% pepsin in sodium citrate buffer, pH 4.5, for 1 hour at 37°C, then dialysed against 0.1 M sodium phosphate, pH 6, with 0.5 mM EDTA and reduced for 1 hour at room temperature using mercaptoethylamine hydrochloride (13.3 mg/mg antibody). F(ab') fragments were separated over a Sephadex PD-10 gel filtration column equilibrated with 20 mM sodiumphosphate with 150 mM NaCl and 1 mM EDTA, pH 6.5, then incubated with threefold molar excess of Monomaleimido Nanogold solution for 18 h at 4°C. Unbound gold particles were separated from antibody conjugates by gel filtration (Superose-12 column).
Cells were incubated with Nanogold-F(ab') fragments in PBS at 4°C for 1 hour, allowed to internalize at 37°C for 10 min, then processed for in vitro vesicle formation and cryoelectron microscopy. The resulting vesicle supernatant was fixed with 3% formaldehyde and 0.2% glutaraldehyde for 2 hours at room temperature and pelleted by centrifugation at 100,000 x g for 1 hour. The pellet was washed with 0.1 M phosphate buffer, pH 7.4. Free aldehyde groups were quenched by incubation in 50 mM NH4Cl for 30 min at room temperature. After three rinses with phosphate buffer, the sample was processed for cryosectioning: the pellet was mixed with 10% gelatin, cooled on ice, cut into small pieces and infiltrated with 2.3 M sucrose overnight at 4°C, frozen in liquid nitrogen on cutting pins, and cryosectioned at -120°C with a Leica Ultracut UCT ultramicrotome. Sections were thawed and transferred to Formvar-coated nickel grids. The Nanogold marker was enhanced by silver (HQ Silver enhancement kit; Nanoprobes). AP-1 complexes were localized with monoclonal mouse anti-gamma-adaptin antibodies followed by 10 nm gold-labeled goat anti-mouse IgG. Grids were stained, dried and viewed in the electron microscope.
Vesicle formation was found to be strongly inhibited upon immunodepletion of adaptor protein (AP)-1, but not of AP-2 or AP-3, from the cytosol, and was restored by readdition of purified AP-1. Vesicle formation was stimulated by supplemented clathrin, but inhibited by brefeldin A, consistent with the involvement of ARF1 and a brefeldin-sensitive guanine nucleotide exchange factor. The GTPase rab4, but not rab5, was required to generate endosome-derived vesicles. Depletion of rabaptin-5/rabex-5, a known interactor of both rab4 and gamma-adaptin, stimulated vesicle formation while addition of the purified protein strongly inhibited it. These results indicate that recycling is mediated by AP-1/clathrin-coated vesicles, and regulated by rab4 and rabaptin-5/rabex-5.
Reference:
Pagano, A.; Crottet, P.; Prescianotto-Baschong, C., and Spiess, M.: In vitro formation of recycling vesicles from endosomes requires adaptor protein-1/clathrin and is regulated by rab4 and the connector rabaptin-5.
Mol. Biol. Cell, 15, 4990-5000 (2004).
More information:
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Gold Lipids, Gold-Decorated Layers, and Liposomes
Lipids are a class of compounds with the ability to organize into a wide variety of supramolecular structures. When the lipids are linked to gold particles, these structures may act as templates for the supramolecular assembly of gold particles into useful structures. Nanogold® and undecagold-labeled lipids are a unique class of compounds available from Nanoprobes which are particularly readily used in this manner. Scanning transmission electron microscopy indicated that liposomes formed using gold-labeled lipids can adopt a range of morphologies depending upon the proportion of gold-labeled lipid and the formation conditions, including multi-layer concentric liposomes, micelles, and sheets.
They have also been successfully used for tagging liposomes for microscopic localization and identification. One such application of these probes was the light and electron microscopic tracking of liposomal drug delivery. Nanogold-labeled liposomes containing an anti-fungal channel-forming drug, Ambisome, were shown to pass through the cell wall and enter the cytoplasm, while those without the drug did not:
![[Schematic: pathways of drug and non-drug spiked liposomes in fungal cells] (27k)]](../Images/catfig8.gif)
Schematic showing the pathways of Nanogold-labeled liposomes
in cells, as revealed by EM. Ambisome is a channel-forming anti-fungal
drug: in its absence, the pattern of the label as revealed by silver
enhancement shows that the liposome fuses with the cell membrane
(left), but when it is present, the liposomes enter the cell (right)
and Nanogold accummulates in the cytoplasm.
References:
- Hainfeld, J. F.; Furuya, F. R., and Powell, R. D.: Metallosomes. J. Struct. Biol., 127, 152-160 (1999).
- Adler-Moore, J.: AmBisome targeting to fungal infections. Bone Marrow Transplantation, 14, S3-S7 (1994).
Zhou and co-workers now report the preparation of Langmuir-Blodgett films of organized gold nanoparticles, using octadecylamine-coated gold particles. Octadecylamine-coated gold particles were filtered, washed and dried, then dissolved into chloroform. Methanol was then added to as a poor solvent to selectively dissolve particles with narrower size distribution, giving particles with a 6.8 nm mean diameter. About
400 microliters of 0.5 mg/mL of particles in chloroform was spread on the water surface on a Langmuir-Blodgett trough with a 100 microliter syringe. After the solvent volatilized, the particles were compressed and expanded from 5 to 10 mN/m for several cycles, and the resulting monolayer sequentially transferred onto a pre-hydrophobilized quartz substrate by a vertical LB technique. A multilayer structure was created layer by layer by the LangmuirBlodgett method. Transmission electron microscopy images and small-angle X-ray diffraction respectively confirmed that the particles were well-ordered within each monolayer and aligned from layer to layer. One point though: 6.8 nm colloidal gold is not Nanogold; the name is a trademark of Nanoprobes, and applies only to our products.
Reference:
Zhou, X.; Liu, C.; Jiang, L., and Li, J.: Formation of an ordered passivated-nanogold multilayer by the LangmuirBlodgett method. Coll. Surf. A.: Physicochem. Eng. Aspects, 248, 43-45 (2004).
More information:
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Nanoprobes at the San Antonio Breast Cancer Symposium
In collaboration with Dr. Raymond R. Tubbs of the Cleveland Clinic Foundation, we will be presenting a poster at the San Antonio Breast Cancer Symposium (December 8 - 11 at the Henry B. Gonzalez Convention Center, San Antonio, TX) on the validation of SILVERFISH, a novel brightfield assay for HER2. Using SILVERFISH, both the amplification of the HER2 oncogene and overexpression of the HER2 protein are visualized simultaneously, using enzyme metallography in situ hybridization, and Fast Red K immunohistochemistry. Look for poster # 5031 in poster session 5, which will be held on Friday December 10 at 4:30 - 7:00 pm in Exhibit Hall B
More information:
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Other Recent Publications
What else, besides Nanogold and organic quenchers, effectively quenches molecular beacons? Copper (II) complexes, according to Brunner and Kraemer in their report in the Journal of the American Chemical Society. A commercially available, 3'-fluorescein, 5'-aminolink 25mer DNA was functionalized at the 5'-amino position with carboxy-functionalized 5-(2-pyridinyl)pyrazole and treated with Cu(II) ions. Preliminary experiments indicated signal-to-noise ratios of 15, comparable to organic quenchers.
Reference:
Brunner, J., and Kraemer, R.: Copper(II)-quenched oligonucleotide probes for fluorescent DNA sensing. J. Amer. Chem. Soc., 126, 13626-13627. (2004).
A new isothermal signal amplification method for DNA and RNA was described in the same journal by Abe and Kool. They applied a strategy in which two "self-ligating half-probe" oligonucleotides hybridize to a target, then react to form the complete probe by means of complementary reactive groups built into their ends. One half-probe is labeled with a fluorophore and a DABCYL group. The DABSYL group, a widely used fluorescence quencher, acts as the leaving group in the reaction: therefore, after reaction the probe has a fluorescent signal that is used for detection. Abe and Kool use a novel class of DABSYL cross-linkers, attached via an alkane chain, that result in destabilization of the probe-target hybrid once the link is made, promoting dissociation and driving turnover and signal amplification. This resulted in up to 92-fold amplification, and a reaction rate 4-5 times faster than previously reported approaches to ligation-based signal amplification.
Reference:
Abe, H., and Kool, E. T.: Destabilizing universal linkers for signal amplification in self-ligating probes for RNA. J. Amer. Chem. Soc., 126, 13980-13986 (2004).
Yu and Yam have added an article on how to prepare 50 nm silver nanocubes to the growing literature on controlling the morphology of nanoparticles. Monodisperse silver nanocubes with a mean edge length of 55 ± 5 nm were prepared by a HTAB (n-hexadecyltrimethylammonium bromide) - modified silver mirror reaction at 120°C. The silver mirror reaction is an "old" chemical route, but introduction of HTAB to the mixture of [Ag(NH3)2]+ (aq) and glucose used for the reaction produces a much more controlled reaction which proceeds at a much higher temperature to yield monodisperse nanocubes. X-ray diffraction confirmed the fcc structure of the silver.
Reference:
Yu, D., and Yam, V. W.: Controlled synthesis of monodisperse silver nanocubes in water. J. Amer. Chem. Soc., 126, 13200-13201 (2004).
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