N A N O P R O B E S E - N E W S
Vol. 7, No. 5 May 11, 2006
Updated: May 11, 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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Correlative Fluorescence and SEM with FluoroNanogold
We have previously described the use of Nanogold® for scanning electron microscopy (SEM), both with in situ hybridization and for correlative light and electron microscopy, using a platinum blue and a Nanogold-antibody conjugate, or Nanogold and immunofluorescence. In a recent issue of Chromosoma, Elizabeth Schröder-Reiter and group now describe the use of the combined fluorescent and gold probe FluoroNanogold, visualized by high-resolution 3D analytical SEM, to show the architecture and DNA distribution a nucleolus organizing region (NOR) with atypical peg-like terminal constriction on metaphase plant chromosomes. Improvements in signal localization, labeling efficiency, and structural preservation in the SEM ISH procedure allowed 3D SEM analysis of the NOR structure and rDNA distribution for the first time.
An atypical, peg-like terminal constriction ("peg") on metaphase chromosomes of the plant genus Oziroë was identified as a nucleolus organizing region (NOR) by detecting 45S rDNA with correlative light microscopy (LM) and scanning electron microscopy (SEM) in situ hybridization (ISH).
Oziroë chromosomes were isolated and mounted on laser-marked glass slides using the drop/cryo technique (Martin, R.; Busch, W.; Herrmann, R. G., and Wanner, G: Efficient preparation of plant chromosomes for high-resolution scanning electron microscopy. Chromosome Res., 2, 411415 (1994)). Air-drying was strictly avoided: this was found to be essential for the preservation of 3D chromosome ultrastructure and for viewing access of the metaphases with SEM. For exclusive SEM analysis, chromosome slides were subsequently fixed in 2.5% (v/v) glutaraldehyde in cacodylate buffer (75 mM, pH 7); for chromosome slides for in situ hybridization, control slides for exclusive LM evaluation were air-dried after the drop/cryo procedure. Slides for correlative LM and SEM were incubated for 30 minutes in 2.5% glutaraldehyde in saline sodium citrate (SSC) buffer (pH 7.2). Phase contrast images of metaphase spreads were photographed with fixative and coverslips. For separate visualization of DNA in SEM, chromosomes were stained for 30 minutes at room temperature with platinum (Pt) blue ([CH3CN]2Pt oligomer, 10 mM, pH 7.2) and washed with distilled water and then with 100% acetone prior to critical point drying.
For in situ hybridization, a plasmid VER17 encoding part of the 18S, the 5.8S, most of the 25S, and the internal transcribed spacers of Vicia faba 45S rRNA, was used as an rDNA-specific probe. 45S rDNA was labeled by nick 51 translation with biotin-16-dUTP, and for ISH, 20 ng probe was applied per slide. For correlative LM and SEM ISH, the procedure was shortened by omitting the enzymatic (pepsin, RNase) treatment and intermediate dehydration steps, and to ensure structural preservation of the chromosomes, it was essential that all air-drying steps were avoided. Prior to detection of biotin-labeled probes, slides were incubated in a blocking solution (5% bovine serum albumin in 4 standard sodium citrate (SSC) with 0.2% Tween-20) for 30 minutes at 37°C. Alexa Fluor®* 488 FluoroNanogoldstreptavidin, diluted at 1:100 in 1% bovine serum albumin in 4 SSC with 0.1% Tween 20), or fluorescein isothiocyanate (FITC)streptavidin was then applied, incubated for 1 hour in a moistened, light-protected chamber at 37°C, and washed 35 min in 2 SSC. Specimens were mounted in 1% (w/v) 4',6-diamidino-2-phenylindole (DAPI) dissolved in Vectashield (Vector, Burlingame, CA, USA), slides examined, and fluorescent images digitally recorded.
After specimens were evaluated by fluorescence microscopy, coverslips were removed by floating in 100% ethanol (EtOH). Specimens were washed three times with 100% ethanol to remove the Vectashield, washed with distilled water, and silver-enhanced for 5 minutes using HQ Silver. After a final wash series in distilled water, slides were washed in 100% acetone, critical point dried from CO2, checked by phase contrast light microscopy, cut to size with a glass cutter, and mounted with double-sided tape to an aluminum stub with conductive carbon cement. Unlabeled specimens were sputter coated with platinum to a layer of approximately 35 nm and examined at accelerating voltages between 8 and 15 kV; labeled specimens (with Nanogold or with Pt blue) were carbon-coated by evaporation to a layer of 35 nm and examined at either 25 or 30 kV. Backscattered electrons (BSE) were detected with a YAG-type detector (Autrata). Secondary electron (SE) and BSE images were recorded simultaneously.
High-resolution 3D analytical SEM showed that the architecture and DNA distribution of the peg-like NOR were typical for chromosomes, although the chromomeres were significantly smaller. Improvements to the SEM ISH procedure enabled more accurate signal localization, higher labeling efficiency, and better structural preservation, and for the first time this allowed 3D SEM analysis of the peg-like NOR structure and rDNA distribution. FluoroNanogold proved to be an attractive tool that allows efficient immunodetection in both LM and SEM. Based on the data obtained, a model was proposed for the peg structure and its mode of condensation.
Schröder-Reiter, E.; Houben, A.; Grau, J, and Wanner, G.: Characterization of a peg-like terminal NOR structure with light microscopy and high-resolution scanning electron microscopy. Chromosoma, 115, 50-59. (2006).
Alexa Fluor®* 488 FluoroNanogold conjugates may be observed by fluorescence microscopy using the same filter set used for fluorescein conjugates, so you do not need to change your observation procedure. Furthermore, if you then use Alexa Fluor®* 594, you can differentiate a second FluoroNanogold-labeled target from a feature labeled with fluorescein, Alexa Fluor® 488, green fluorescent protein, or other green fluorophores. The combined Alexa Fluor®* and gold probes offer superior performance levels and additional features:
- Increased fluorescence brightness and higher quantum yield.
- Improved solubility: lower background signal and higher signal-to-noise ratios.
- Fluorescence remains high and consistent across a wider pH range.
- Uses fluorescein (Alexa Fluor®* 488) or Texas Red (Alexa Fluor®* 594) filter sets.
Alexa Fluor® 488 FluoroNanogold: (left) Structures Alexa Fluor® 488 FluoroNanogold-Fab' and streptavidin conjugates, showing separate covalent attachment of the two labels. (right) Fluorescent staining obtained using combined combined Alexa Fluor®* 488 and Nanogold® - Fab' tertiary probe. The specimen is a slide from the NOVA Lite ANA HEp-2 test, an indirect immunofluorescent test system for the screening and semi-quantitative determination of anti-nuclear antibodies (ANA) in human serum, stained using positive pattern control human sera, Mouse anti-Human secondary antibody, and Alexa Fluor® 488 FluoroNanogold - Fab' tertiary probe. Specimens were washed with PBS (30 minutes) between each step, then blocked by the addition of 7% nonfat dried milk to the tertiary antibody solution (original magnification 400 X).
* Alexa Fluor is a trademark of Molecular Probes / Invitrogen
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Gold enhancement is an autometallographic method, similar to silver enhancement, in which gold is deposited onto gold nanoparticles. It has significant advantages for both scanning electron microscopy (SEM) and transmission electron microscopy (TEM):
- Gold enhancement may safely be used before osmium tetroxide - it is not etched.
- May be used in physiological buffers (chlorides precipitate silver, but not gold).
- The metallographic reaction is less pH sensitive than that of silver.
- Gold gives a much stronger backscatter signal than silver.
- GoldEnhance is near neutral pH for best ultrastructural preservation.
- Low viscosity, so the components may be dispensed and mixed easily and accurately.
In addition to Kenzaka, other users are discovering the benefits of our gold enhancement technology. Alexander and co-workers, in their recent paper in Proceedings of the National Academy of Sciences of the USA showed that gold enhancement works as well with conventional colloidal gold as it does with Nanogold®: they used a combination of immunofluorescence microscopy and SEM with gold-enhanced colloidal gold labeling to study the distribution and mobility of the sodium-hydrogen exchanger isoform 3 (NHE3), located on the apical membrane renal epithelial cells, which is fundamental to the maintenance of systemic volume and pH homeostasis. Using a combination of fluorescence microscopy followed by immunoelectron microscopy using gold-enhanced 18 nm colloidal gold, they concluded that modulation of the mobile fraction of NHE3 on the apical membrane can alter the number of functional exchangers on the cell surface and hence the rate of transepithelial ion transport; regulation of the interaction of NHE3 with the actin cytoskeleton may therefore provide a new mode of regulation of sodium and hydrogen transport.
Gold-enhanced 10 nm colloidal gold was recently used by Liu, Meagher and group to visualize the distribution of vimentin in whole mounts by transmission electron microscopy and scanning electron microscopy as part of a study to demonstrate that the follicle-associated epithelium of the rabbit conjunctiva contains a cell with the same morphologic characteristics and the ability to bind and translocate latex beads as M cells; these were indistinguishable from antigen sampling M cells in the rabbit cecum and tonsils.
Liu, H.; Meagher, C. K.; Moore, C. P., and Phillips, T. E.: M cells in the follicle-associated epithelium of the rabbit conjunctiva preferentially bind and translocate latex beads. Invest. Ophthalmol. Vis. Sci., 46, 4217-4223 (2006).
In some situations, however, GoldEnhance may produce a fine, "granular" non-specific background signal of small particles. There are several ways in which you can either slow down development so that you have more control over its progress, or chemically "stop" the development process. Which approach is best for the system under study depends upon whether you believe the primary problem is continued development after rinsing, in which case a chemical "stop" is appropriate, or excessively fast development before rinsing, in which case slowing down the reaction is most appropriate.
If you are observing fine background that seems to arise from continued development after rinsing to remove the GoldEnhance reagents, a number of "stop" procedures are available. The simplest and most universal is treatment with 1% or 2% freshly prepared sodium thiosulfate for a few seconds, and unless there are specific reasons that this would be harmful to your specimen, we usually suggest that you try this first.
A number of other specific methods have been reported in the literature for stopping silver enhancement. Since gold enhancement is a chemically similar process, these reactions are also applicable to gold enhancement:
- 1% acetic acid (Reference: Scopsi, L.: Silver-enhanced colloidal gold method. In: Colloidal Gold: Principles, Methods, and Applications; M. A. Hayat (Ed.), Vol. 1, p. 260. Academic Press, San Diego, CA (1989)).
- 1% acetic acid followed by photographic fixer (Agefix, Agfa-Gevaert, or Ilfospeed 200, Ilford) (Reference: Scopsi, L.: Silver-enhanced colloidal gold method. In: Colloidal Gold: Principles, Methods, and Applications; M. A. Hayat (Ed.), Vol. 1, p. 260. Academic Press, San Diego, CA (1989)).
- Direct photo fix, using those just mentioned (Reference: Burry, R. W.: Pre-embedding immunocytochemistry with silver-enhanced small gold particles, p. 217-230. In: Immunogold silver staining: Principles, methods and applications; M. A. Hayat (Ed.). CRC Press, Boca Raton, FL (1995)).
- Brief rinse in 2.5% sodium chloride (Reference: Scopsi, L.: Silver-enhanced colloidal gold method. In: Colloidal Gold: Principles, Methods, and Applications; M. A. Hayat (Ed.), Vol. 1, p. 260. Academic Press, San Diego, CA (1989)).
- 15-25% aqueous sodium thiosulfate plus 15% sodium sulfite.
Danscher, G.: Histochemical demonstration of heavy metals. A revised version of the silver sulphide method suitable for both light and electron microscopy. Histochemistry, 71, 1-16 (1981).
- 1% acetic acid, washes in acetate buffer, toning in 0.05% HAuCl4 for 3-10 minutes, with excess silver removed with 3% sodium thiosulfate. Nanogold-labeled proteins run on a polyacrylamide gel have been found to produce low backgrounds when stopped with 10% acetic acid with 10% glucose in water, compared with just water rinsing to stop development.
Takizawa, T., and Robinson, J. M.: Use of 1.4-nm immunogold particles for immunocytochemistry on ultra-thin cryosections. J. Histochem. Cytochem., 42, 1615-1623 (1994).
If you believe that your principal problem is that the reaction is too fast and you need to slow down development, the following modifications are recommended. We suggest trying (1), (2) or (3) first:
- Reduce the development time, to one minute. The specific signal may be sufficiently dense even at the shorter development times, so reducing the time further may remove the background while still giving a strong specific signal.
- When mixing and dispensing the GoldEnhance, use a mixture of 5 parts solution B (activator) to one part solution A (enhancer). Solution B contains a gold stabilizing agent to control the reactivity of gold in solution: by increasing the amount of B, you may inhibit the background while still permitting strong specific deposition.
- Substitute Solution D with 0.05M sodium phosphate with 0.1M sodium chloride, at pH 5.5. This will reduce the pH of the reaction mixture. Generally, autometallographic reactions (silver and gold enhancement) proceed more slowly at lower pH; b reducing the pH, you will slow the gold deposition reaction and gain more control.
- Increase the sodium chloride concentration in your substitute for D to 0.5M. We have occasionally observed that this reduces background (possibly by shortening the range of ionic interactions in the solution through higher ionic strength).
- Treat samples with Lugol's iodine (Sigma: 30 seconds) followed by 1% sodium thiosulfate (30 seconds) before application of the antibodies- this reagent is used to remove heavy metals from tissue slides for light microscopy, and since these can be catalysts for autometallographic development, this eliminates one source of background. We have found it to be essential for in situ hybridization detection with Nanogold and GoldEnhance. In the in situ hybridization procedure it is applied early, before the Nanogold reagent, so if it is possible it may be best to try it before adding the antibodies.
- Add a viscosity modifier, such as polyethylene glycol (carbowax, or one of the higher MW polethylene glycols, would be best: dissolve 4% in your substitute solution D to give 1% in the final mixture). Gum Arabic, if available, is also highly effective in silver enhancement solutions, although it must be allowed to dissolve for 24 hours before use: use 30% in solution D (make up a 60% solution, then mix with an equal volume of a double concentration of your substitute solution D). These slow down development in silver enhancement to give a very controlled, homogeneous rate of reaction, resulting in uniform particle size and morphology.
- Add a small amount of detergent, such as 0.1% Tween-20 (add as 0.4% in your substitute D to give 0.1% in the final solution). Detergents are known to modify the growth of nanoparticles, and act to negate hydrophobic interactions which can initiate background deposition, or facilitate the deposition of any pre-formed gold clusters from solution.
- Wash with 0.6 M triethylammonium bicarbonate buffer in 20 % isopropanol/water after gold enhancement. We find this buffer to be highly effective for dissolving small gold particles such as Nanogold, and it will help to remove any particulate deposits. It is prepared by bubbling carbon dioxide through a mixture of degassed water and degassed triethylamine. We recommend making a 2 M stock solution: dilute in water/isopropanol to give 0.6 M in 20 % isopropanol/water.
Reference for preparation:
Safer, D.; Bolinger, L., and Leigh, J. S.: Undecagold clusters for site-specific labeling of biological macromolecules: simplified preparation and model applications. J. Inorg. Biochem., 26, 77-91 (1986).
Wanzhong He, working in the laboratory of Dr. Pamela J. Bjorkman in the Division of Biology at the California Institute of Technology has been kind enough to send us feedback on on the results of applying these methods. A combination of steps was used to eliminate background:
- A ratio of one part solution A (enhancer) : 5 parts solution B (activator) was used.
- Solution D (buffer) was substituted with 0.05 M sodium phosphate with 0.1 M sodium chloride, pH 5.5.
- Enhancement was performed for 10-15 minutes rather than 3 minutes.
- The reaction was "stopped" using 3% Na2S2O3 for 30 seconds, then washed with 10% acetic acid + 10% glucose for 3 x 5 minutes.
Some preliminary images are shown below:
left: Gold enhancement of intestinal tissue with Nanogold staining. (a) Nanogold particles, then enhanced with GoldEnhance EM for 3 minutes; (b) control with Nanogold labeling omitted, showing the granular background. right: analogous procedure, using modified protocol to control background, comprising: one part A (enhancer) : 5 parts B (activator); D (buffer) was substituted with 0.05M sodium phosphate with 0.1M sodium chloride, at pH 5.5; enhancement was performed for 10-15 minutes rather than 3 minutes, "stopped" using 3% Na2S2O3 30 seconds, then washed with 10% acetic acid + 10% glucose for 3 x 5 minutes. (c) Nanogold particles, enhanced with GoldEnhance EM; (d) control with Nanogold labeling omitted, identical gold enhancement procedure. Insets show experimental (left) and control (right) specimens (Thanks to Wanzhong He and Dr. Pamela Bjorkman for the images).
If none of these are effective, you might consider switching to silver enhancement using LI Silver or HQ Silver instead. If you are concerned about osmium etching of deposited silver, it is worth noting that Burry and co-workers find that this may frequently be avoided by using 0.1 % osmium tetroxide; this still gives excellent staining, but etching is no longer a problem (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 p. 217-230 (1995)).
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Everts and group report a novel application of Nanogold® labeling in their recent paper in Nano Letters: labeling the adenovirus gene therapy vector in order to target gold to cancer cells for near-infrared thermal therapy, expanding the recently reported applications of gold nanpoparticles as X-ray contrast agents and radiotherapy enhancers. The authors conducted a straightforward labeling procedure using Mono-Sulfo-NHS-Nanogold to label the amino- side-chains of lysine residues in the adenovirus capsid.
Nanogold® Labeling of Adenovirus: Mono-Sulfo-NHS-Nanogold, which reacts specifically with amino- groups, was used to label the viral capsid at lysine residue sites; there are more than 10,000 lysines in the capsid proteins, although not all are accessible for labeling.
The adenoviral vector encoding firefly luciferase (Luc) under transcriptional control of the constitutively active cytomegalovirus (CMV) promoter, AdCMVLuc, was constructed and used for this project. Primary amines are abundantly present on the adenoviral capsid, which contains more than 10,000 lysine residues, although not all are accessible for labeling. Mono-Sulfo-NHS-Nanogold, a 1.4 nm gold nanoparticle containing a peripheral amine-reactive sulfo-N-hydroxy succinimide group, was dissolved in DMSO to a final concentration of 200 fM immediately before reaction with the adenoviral vectors. Varying amounts of gold were added to a fixed amount of AdCMVLuc, diluted in 10 mM HEPES buffer containing 10% glycerol and 1 mM MgCl2, pH 7.8. Ratios of gold to adenovirus used in syntheses were 100 : 1, 1000 : 1, 3000 : 1 and 5000 : 1 (particle : particle). Gold nanoparticles were allowed to react with adenoviral vectors for 2 hours at room temperature, and the reaction mixture was subsequently stored at 4°C until use.
To determine whether gold was covalently coupled to the adenoviral vector, reaction mixtures were purified using a cesium chloride (CsCl) gradient, with fractions collected from the bottom of the centrifugation tube. Fractions were analyzed for the presence of adenovirus using slot blot staining for the hexon capsid protein, and the presence of gold using slot blot staining with silver enhancement. Hexon staining was performed using a polyclonal goat anti-hexon antibody followed by an HRP-conjugated rabbit anti-goat secondary antibody: signal was detected using Western Lightning chemiluminescence reagent (Perkin-Elmer Life Sciences) recorded on film. Gold staining was visualized using LI Silver enhancement. a comparison of the staining patterns of both slot blots demonstrated colocalization of virus with gold, indicating co-migration of both components through the CsCl gradient and suggesting a covalent association between the Nanogold and adenoviral vectors. During centrifugation, a shift in the height of the viral band in the centrifuge tube was observed: this was dependent on the excess of Nanogold used in the synthesis procedure, indicating an increased density of the viral particles upon gold labeling. To determine whether labeling was specifically due to the covalent reaction of the gold particles with amino- groups, a control experiment was conducted, in which the viral vectors were mixed with NTA-Ni(II)-Nanogold instead of Mono-Sulfo-NHS-Nanogold: no labeling was observed.
Gold-labeled adenoviral vectors were examined by electron microscopy at 80 kV. Vectors were deposited onto carbon-coated copper grids; no staining was used, in order to avoid occlusion of the 1.4 nm nanoparticles on the surface of the virions. Gold nanoparticles were not observed in unlabeled or NTA-Ni(II)-Nanogold-labeled virus preparations. In contrast, gold nanoparticles could clearly be observed in the virus preparation labeled with sulfo-NHS gold nanoparticles as small black dots on the edges of the virions, and showed sharp boundaries and similar sizes.
To investigate whether gold nanoparticle attachment might result in a loss of infectivity of the adenoviral vectors, gene transfer was evaluated using the luciferase encoding adenoviral vector AdCMVLuc, labeled with different amounts of gold, in HeLa cells known to be readily infected with adenoviral vectors. AdCMVLuc infectivity at a multiplicity of infection (MOI) of 25 was not affected by the synthesis procedure itself, as demonstrated by the comparable levels of luciferase activity of sham-labeled versus fresh, unmodified AdCMVLuc. With a gold : adenovirus ratio of 100 : 1 (particle : particle), there was no effect on affect infectivity. Higher gold : adenovirus ratios did decrease infectivity compared with unlabeled AdCMVLuc. Results were similar for lower (5) and higher (125) MOIs. This suggests a threshold of the number of gold nanoparticles that can be coupled to adenoviral vector without disrupting the natural infectivity mechanism of gene transfer.
These results demonstrate that covalent coupling of gold nanoparticles to adenovirus could be achieved, while retaining virus infectivity and ability to retarget tumor-associated antigens. This supports the use of adenoviral vectors as carriers for gold nanoparticles, and also suggests that a single-agent combination gene and gold nanoparticle hyperthermia therapy is possible in which the two therapeutic agents are delivered by the same carrier.
Everts, M.; Saini, V.; Leddon, J. L.; Kok, R. J.; Stoff-Khalili, M.; Preuss, M. A.; Millican, C. L.; Perkins, G.; Brown, J. M.; Bagaria, H.; Nikles, D. E.; Johnson, D. T.; Zharov, V. P., and Curiel, D. T.: Covalently Linked Au Nanoparticles to a Viral Vector: Potential for Combined Photothermal and Gene Cancer Therapy. Nano Lett., 6, 587-591 (2006).
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Inhibition of Insulin Fibrillogenesis: Using Nano-W to Study Aggregation
Negative stains are used to define the edges of particulate or suspended specimens with low contrast, such as protein complexes or viruses, for electron microscopy. They are particularly important for high-resolution structural studies of viruses and other macromolecular protein structures with a defined assembly pattern, where visualization of the entire structure and its orientation is required, such as for image analysis, rather than the localization of a specific site by gold labeling.
The ideal negative stain is amorphous, since crystallization can obscure features of interest. When used with ultrastructural gold labeling, it is helpful if it is not too electron-dense, so that contrast is preserved between the gold particles and their environment. Nanoprobes offers two novel negative staining reagents, NanoVan and Nano-W, based on vanadium and tungsten respectively, that allow negative staining with a range of different densities. NanoVan is recommended for use with Nanogold® because the lower atomic number of vanadium means that the stain is less electron-dense than heavy metal-based stains such as uranyl acetate or lead citrate and allows easier visualization of the Nanogold particles. It is very fine-grained and highly amorphous, and has been used for a number of high-resolution STEM and TEM studies of virus and protein ultrastructure. Nano-W gives a more dense stain, and is more suited to use with larger gold labels.
Advantages of these reagents:
- NanoVan and Nano-W are completely miscible: they may be mixed in different proportions to give any 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.
Gibson and co-workers demonstrate that negative staining also provides a useful tool for studying protein aggregation: in their recent paper in Protein Science, they use it to evaluate the effects of a specifically targeted peptide upon insulin aggregation. At acidic pH and elevated temperature, insulin partially unfolds and aggregates into highly structured amyloid fibrils. Insulin aggregation means loss of activity, and can trigger an unwanted immune response. Compounds that prevent protein aggregation have been used to stabilize insulin: however, these generally suppress aggregation only at relatively high inhibitor concentrations and excesses: 100 mM or greater arginine concentrations are required to effectively inhibit aggregation of 0.5 mM insulin, for example. As an alternative strategy, the authors used small hybrid peptides, consisting of a recognition domain designed to specifically bind to insulin, and a disrupting domain that alters insulin aggregation. Proteolytic fragments of insulin were screened to identify short peptides as putative recognition domains. Hybrid peptides were then synthesized by appending a hexameric-arginine disrupting domain to the identified recognition domain.
Hybrids constructed in this manner were evaluated for their ability to stabilize insulin at pH 2.0 and 37&176;C, conditions that normally lead to partial unfolding and amyloidogenesis. Amyloid fibril formation was quantified using a thioflavin T (ThT) fluorescence assay, in which measured ThT fluorescence intensity is taken to be proportional to the mass of amyloid fibrils. After an initial lag phase of 4555 h, during which ThT fluorescence remained at background levels, a rapid increase in fluorescence intensity was observed. This coincided with the appearance of macroscopic aggregates. Using dynamic light scattering, no aggregate growth was observed for 50 hours, followed by the sudden formation of very large aggregates. Transmission electron microscopy was then used to confirm that the aggregates had fibril morphology. Aggregated samples used for ThT experiments were collected when visible precipitate first appeared, and then stored for 2 weeks at room temperature. These samples were centrifuged at 16,000g for 5 min and most of the supernatant was discarded. Deionized water (50 mL) was added to the remaining pellet, and the samples were vortexed rapidly for 20 seconds, then stained with Nano-W (methylamine tungstate) and placed on a pioloform coating grid support.
Data were analyzed using a simple two-step model of aggregation kinetics. Of the proteomic fragments investigated, the short peptide VEALYL, corresponding to residues B1217 of full-length insulin, was found to interact most with full-length insulin; therefore a hybrid peptide was synthesized that contained this binding domain combined with and hexameric arginine. The target peptide significantly reduced the rate of insulin aggregation at near equimolar concentrations. An effective binding domain and N-terminal placement of the arginine hexamer were found to be necessary for inhibitory activity. These results are useful not only in identifying a specific insulin aggregation inhibitor, but also in validating a targeted protein strategy for modifying the aggregation of amyloidogenic proteins.
Gibson, T. J., and Murphy, R. M.: Inhibition of insulin fibrillogenesis with targeted peptides. Protein Sci., 15, 1133-1141 (2006).
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Price Adjustment: Order by May 31 to Take Advantage of Current Prices
Our goal at Nanoprobes is to provide you with the most effective means of finding the research answers that you seek. Our constant research and development has resulted in additional unique products and enhancements, and we look forward to additional new products shortly. We work consistently to keep prices affordable and competitive, and we have maintained our current price schedule since May 2000. However, rising costs and prices for our supplies and materials mean we now need to raise the prices slightly on most of our product lines, effective June 1. Therefore, if you use our products and need to make your funds go further, we encourage you to place your orders before May 31 so that you can take advantage of our current prices.
Thank you for using Nanoprobes as your partner in research. We will continue to provide our valued customers with superior products and complete, timely technical support. Please keep reading our Newsletter for new developments and product releases planned for the coming months.
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Other Recent Publications
Pre-embedding labeling with HQ Silver enhancement for immunoelectron microscopy remains the most popular application of our Nanogold® conjugates, and Inamura and colleagues contribute a new paper on this method in the current issue of Neuroscience Research. They used both pre-embedding immunoelectron microscopy and Sodium dodecyl sulfate (SDS)-digested freeze-fracture replica labeling (SDS-FRL) with 5 and 10 nm colloidal gold to localize the transmembrane AMPA (alpha-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate) receptor regulatory proteins (TARPs) stargazin/g-2 and g-8 in rat coronal brain sections. Pre-embedding immunoelectron microscopy was carried out using a modified pre-embedding Nanogold-silver enhancement method: 40 µm sagittal sections were blocked with 20% Block Ace (Dainihonseiyaku, Osaka, Japan) in PBS with 0.02% saponin for 10 minutes, incubated with either rabbit anti-stargazin/g-2 (ct) or anti-g-8 (ct) antibody in PBS containing 5% Block Ace and 0.005% saponin for 48 hours at 4°C, then washed with 5% Block Ace in PBS containing 0.005% saponin (3 x 20 minutes). Sections were then incubated with a Nanogold-Fab' Goat anti-Rabbit fragments at a dilution of 1:50 for 24 h at 4&176;C, then fixed in 1% glutaraldehyde for 10 minutes. After washing in 50 mM HEPES buffer, pH 5.8 (2 hours), bound Nanogold particles were enhanced by incubation with HQ silver at 20°C for 12 minutes in the dark. Sections were postfixed with 0.5% osmium tetroxide in phosphate buffer (pH 7.4) for 1 hour, dehydrated using a graded series of ethanol and propylene oxide, and embedded in Epon 812. Ultrathin sections were cut, stained with uranyl acetate and lead citrate, and observed by transmission electron microscopy. Analysis of sodium dodecyl sulfate-digested freeze-fracture replica labeling revealed that stargazin/g-2 was concentrated in the postsynaptic area, whereas g-8 was distributed both in synaptic and extra-synaptic plasma membranes of the hippocampal neuron. Upon separation of a Triton X-100-treated synaptic plasma membrane-enriched brain fraction by sucrose density gradient ultracentrifugation, a large proportion of NMDA (N-methyl-D-aspartate) receptor and stargazin/g-2 was accumulated in raft-enriched fractions, whereas AMPA receptor and g-8 were distributed in both the raft-enriched fractions and other Triton-insoluble fractions. Phosphorylation of stargazin/g-2 and g-8 was regulated by different sets of kinases and phosphatases in cultured cortical neurons. These results suggest that stargazin/g-2 and g-8 have distinct roles in postsynaptic membranes under the regulation of different intracellular signaling pathways.
Inamura, M.; Itakura, M.; Okamoto, H.; Hoka, S.; Mizoguchi, A.; Fukazawa, Y.; Shigemoto, R.; Yamamori, S., and Takahashi, M.: Differential localization and regulation of stargazin-like protein, gamma-8 and stargazin in the plasma membrane of hippocampal and cortical neurons. Neurosci. Res.,, 55 45-53 (2006).
Original reference for pre-embedding method:
Mizoguchi, A.: Rab3A-Rab GDI-Rabphilin-3A system regulating membrane fusion machinery in the synapse and growth cone. Acta Histochem. Cytochem., 27, 117126 (1994).
Jennings, Singh and Strouse expand our knowledge of energy transfer interactions between gold nanoparticles and fluorophores in their recent article in the Journal of the American Chemical Society. The fluorescence behavior of molecular dyes at discrete distances from 1.5 nm diameter gold nanoparticles as a function of distance and energy is investigated. Gold nanoparticles were synthesized by rapid reduction of hydrogen tetrachloroaurate ([AuCl4]-.3H2O) in the presence of tetraoctylammonium bromide by sodium borohydride in an argon-sparged water/toluene mixture. TEM measurements yielded an average diameter of 1.5 ± 0.5 nm. After washing thoroughly with a variety of solvents, including hexanes, sodium nitrite solution, and methanol-water, ligand exchange was accomplished by stirring ~400 mg bis(p-sulfonatophenyl) phenylphosphine dihydrate in purified water with 32 mg of purified nanocrystal in 32 mL of methylene chloride overnight: nanoparticles were extracted to the aqueous phase through ligand exchange. Complementary DNA strands engineered to minimize secondary structures, one labeled with 5'-Cy5 and the other with a 5'-thiol, were used for fluorescence measurements. 800 pmol of the single-stranded 5'-thiol DNA were deprotected with 50 mM tris(2-carboxyethyl) phosphine hydrochloride (TCEP) in 20 mM PBS buffer, pH 7.5, at room temperature for 30 minutes and desalted on a NAP-5 column, the dye-containing complementary strand (590 pmol) was immediately added, and the strands annealed at 95°C for 2 minutes, cooled to room temperature for 2 minutes then added to 4 nmol of dry, water-soluble nanoparticles (the 1 : 6.7 dsDNA : nanoparticle stoichiometry ensured predominately 1 : 1 binding). Unbound nanoparticles were separated from the dsDNA-nanoparticle conjugate by successive ethanol precipitations. Photoluminescence and luminescence lifetime measurements, both demonstrate quenching behavior consistent with 1/d4, where d is the separation distance from dye to the surface of the nanoparticle. In agreement with the model of Persson and Lang, all experimental data show that energy transfer to the metal surface is the dominant quenching mechanism, and the radiative rate is unchanged throughout the experiment.
Jennings, T. L.; Singh, M. P., and Strouse, G. F.: Fluorescent Lifetime Quenching near d = 1.5 nm Gold Nanoparticles: Probing NSET Validity. J. Amer. Chem. Soc., 128, 5462-5467(2006).
Chithrani and group, meanwhile, have investigated the relationship between particle size and shape and intracellular uptake of gold nanoparticles, and describe their results in Nano Letters. Spherical and rod-shaped nanoparticles with diameters of 14, 30, 50, 74, and 100 nm, and length by width of 40 x 14 nm and 74 x 14 nm, respectively, were prepared in solution; chemical exchange was then used to coat the rod-shaped gold particles with cetyl trimethylammonium bromide (CTAB). HeLa cells were incubated with gold nanoparticles with various sizes and shapes for 6 hours in Dulbecco Minimum Essential Media (DMEM) plus 10% serum, then detached from the Petri dish surface using trypsin, and homogenized: gold concentration was measured by ICP-AES. Gold particles were taken up predominantly by receptor-mediated endocytosis (RME). The kinetics and saturation concentrations were found to be highly dependent upon the physical dimensions of the nanoparticles: for example, uptake half-life of 14, 50, and 74 nm nanoparticles was 2.10, 1.90, and 2.24 h, respectively, and cellular uptake of rod-shaped structures with lower aspect ratio (1 : 3) was greater than for those with a higher aspect ratio (1 : 5). These results provide guidelines for the size and shape of gold nanoparticles that may work best for applications in cellular or biomedical imaging, or in cell-based therapeutics.
Chithrani, B. D.; Ghazani, A. A., and Chan, W. W. C.: Determining the size and shape dependence of gold nanoparticle uptake into mammalian cells. Nano Lett., 6, 662-668 (2006).
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