N A N O P R O B E S E - N E W S
Vol. 10, No. 1 January 31, 2009
Updated: January 31, 2009
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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Our unique combined fluorescent and gold labeled immunoprobe, FluoroNanogold, is proving useful in many applications, and this month features in two new publications, one on tomography of the nucleus and DNA replication sites, the other mapping the involvement of epidermal growth factor receptor (EGFR) in enterocyte anoikis through the dismantling of E-cadherin-mediated junctions. FluoroNanogold is the only immunoprobe available that can provide both fluorescent and gold labeling with a single probe in one labeling procedure. The covalent linkage assures stability; the entire probe is smaller than an IgG molecule, and because these probes do not require stabilization with additional macromolecules, they show the same high penetration and antigen access of Nanogold-Fab' fragments.
These probes contain an antibody Fab' fragment, covalently linked to both the 1.4 nm Nanogold® label and a fluorescent label (currently, a choice of Alexa Fluor®* 488 or 594, or fluorescein, are available; other fluorescent labels are planned). Our Alexa Fluor®* FluoroNanogold probes offer superior fluorescence labeling performance:
- 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.
Since we offer both Alexa Fluor®* 488 and Alexa Fluor®* 594 FluoroNanogold, you can now use these probes to differentiate multiple targets using different colored fluorescence.
Top: Structure of Alexa Fluor 594 FluoroNanogold-Fab' (left) and streptavidin conjugates (right), showing covalent attachment of Nanogold and Alexa Fluor 594 labels. above: Correlative fluorescence and electron microscopic labeling with Alexa Fluor 594-Streptavidin. Localization of caveolin-1a in ultrathin cryosection of human placenta; caveolin 1 alpha is primarily located to caveolae in placental endothelial cells. One-to-one correspondence is found between fluorescent spots (upper right) and caveola labeled with gold particles (lower right). Ultrathin cryosections, collected on formvar film-coated nickel EM grids, were incubated with chicken anti-human caveolin-1a IgY for 30 minutes at 37°C, then with biotinylated goat anti-chicken F(ab')2 (13 µg/mL, 30 minutes at 37°C), then with Alexa Fluor 594 FluoroNanogold-Streptavidin (1:50 dilution, 30 minutes at room temperature). Non-specific sites on cryosections were blocked with 1% milk - 5% fetal bovine serum-PBS for 30 minutes at room temperature (figure courtesy of T. Takizawa, Ohio State University, Columbus, OH).
In their recent paper in Critical Reviews in Oncology / Hematology, Tchélidzé and group review developments in tomographic analysis of the cell nucleus. Approaches that are reviewed include the tomographic analysis of the 3D spatial distribution of pKi-67 using confocal microscopy and electron tomography: FluoroNanogold labeling enabled the authors to compare the 3D reconstructions obtained at different levels of resolution. Human A549 cells were simultaneously fixed and permeabilized for 4 minutes in 3% paraformaldehyde and 1% Triton X-100 diluted in PBS (140 mM NaCl, 6 mM Na2HPO4, 4 mM KH2PO4, pH 7.2), then saturated for 30 minutes in PBS containing 3% bovine ser/um albumin (BSA), 1 mM CaCl2 and 0.5 mM MgCl2. Slides were incubated for 30 minutes in the presence of MM1 antibodies against human pKi-67 diluted 1:50 in PBS containing 1% BSA, 1 mM CaCl2 and 0.5 mM MgCl2, and rinsed three times for 5 minutes in PBS. Biotinylated goat anti-mouse antibody was then applied for 30 minutes and revealed for 15 minutes with streptavidinFluoroNanogold. For electron microscopy, cells were over-fixed for 12 minutes with 1.6% glutaraldehyde in PBS, rinsed in de-ionized water, then enhanced with HQ Silver for 8 minutes. Cells were harvested by scraping, dehydrated in graded alcohols, and embedded in Epikotte 812.
In optical sections, labeling revealed several thin cords localized mainly around the nucleolus and partly along the nuclear envelope or within the nucleoplasm. In transversal sections, labeled protrusions appeared to contact the nuclear envelope, demonstrating an irregular pattern of labeling arranged as a contorted cord around the nucleolus. Simultaneous labeling of DNA with chromomycin A3 demonstrated that nucleolar pKi-67 was co-localized with DNA. The authors then used electron tomography to study pKi-67 in the same cells. Thick sections (0.52 µm) were tilted in a medium voltage electron microscope operated at 250 kV: a tomogram was obtained, and 50 nm digital sections were analyzed. Labeling was organized into different cords, 300 nm in diameter, comprising thinner fibers 50 nm in diameter; a more detailed characterization of these fibers was carried out at higher magnification of the tomogram.
- Tchélidzé, P.; Chatron-Colliet, A.; Thiry, M.; Lalun, N.; Bobichon, H., and Ploton, D.: Tomography of the cell nucleus using confocal microscopy and medium voltage electron microscopy. Crit. Rev. Oncol. Hematol., 69, 127-143(2009).
- Cheutin, T.; ODonohue, M. F.; Beorchia, A.; Klein, C.; Kaplan, H., and Ploton, D.: Three-dimensional organization of pKi-67: a comparative fluorescence and electron tomography study using fluoronanogold. J. Histochem. Cytochem., 51, 1411423 (2003).
Meanwhile, in another paper in the American Journal of Physiology, Gastrointestinal and Liver Physiology, Lugo-Martínez and colleagues used FluoroNanogold with silver enhancement to confirm the role of EGFR in the disruption of adherens junctions during the onset of anoikis (apoptosis triggered by loss of anchorage) in normal enterocytes detached from the basal lamina. Enterocytes of the intestinal epithelium are continually regenerated from precursor cells in crypts; they migrate along villi, and die when they reach the villus apex after 3-4 days. Cell death is thought to occur by anoikis, but the mechanism of this process was poorly understood. Having previously shown that a key event in the onset of anoikis in normal enterocytes detached from the basal lamina is the disruption of adherens junctions mediated by E-cadherin, the authors further investigated the mechanisms underlying this disassembly of the adherens junctions, using confocal and electron microscopy to study localization of EGFR and E-cadherin during the process.
Correlative light and electron microscopy followed the method described in our own paper. Cryosections of intestine were fixed in 4% paraformaldehyde and 0.1% glutaraldehyde, then labeled with anti-EGFR phosphorylated on tyrosine 845, followed by a secondary Alexa Fluor®-488 Fluoronanogold anti-rabbit Fab' conjugate. EGFR labeled cells were located on sections using an epifluorescence microscope. Then sections were then postfixed in 2.5% glutaraldehyde, and a 5 minute darkroom treatment with HQ Silver was used to enhance the Nanogold. Samples were then dehydrated in graded alcohol and embedded in Epon resin (Poly/Bed 812). Ultrathin sections of ~65 nm were counterstained with uranyl acetate (30 minutes at 40°C) and lead citrate (10 minutes at 25°C), then observed using a JEOL CX100 equipped with a Gatan Digital camera.
The authors found that disruption of the junctions occurs through endocytosis of E-cadherin, a process which in turn depends on the tyrosine-kinase activity of the epidermal growth factor receptor (EGFR). Activation of EGFR was detected in detached enterocytes before E-cadherin disappearance. Specific inhibition of EGFR by tyrphostin AG-1478 resulted in E-cadherin and its cytoplasmic partners beta- and alpha-catenin being maintained at cell-cell contacts, and decreased anoikis. EGFR activation was also found in the intestinal epithelium in vivo, in rare individual cells; these cells were found to lose their interactions with the basal lamina. This supports a mechanism in which EGFR is activated as enterocytes become detached from the basal lamina, and this then contributes to the disruption of E-cadherin-dependent junctions, leading to anoikis. This shows that EGFR participates in the physiological elimination of the enterocytes.
- Lugo-Martínez, V. H.; Petit, C. S.; Fouquet, S.; Le Beyec, J.; Chambaz, J.; Pinon-Raymond, M.; Cardot, P., and Thenet, S.: Epidermal growth factor receptor is involved in enterocyte anoikis through the dismantling of E-cadherin-mediated junctions. Am. J. Physiol., Gastrointest. Liver Physiol., 296, G235-G244 (2009).
- Powell R. D.; Halsey C. M.; Spector D. L.; Kaurin S. L.; McCann J., and Hainfeld, J. F.: A covalent fluorescent-gold immunoprobe: simultaneous detection of a pre-mRNA splicing factor by light and electron microscopy. J. Histochem. Cytochem., 45, 947-956 (1997).
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Our first choice for labeling IgG with Nanogold® is to use Monomaleimido Nanogold to label at a hinge thiol. However, there are a number of other approaches which may be better for your application, and if you require labeling at a different site, have very limited amounts of antibody, or need a rapid procedure, one of the alternatives may be better. Two alternative Nanogold labeling strategies which may be conducted readily using available reagents are oxidizing a sugar residue in the glycosylated Fc region of the IgG and labeling with Monoamino Nanogold, and labeling at an amino- site (either an N-terminal amine or a lysine residue) using Mono-Sulfo-NHS-Nanogold. These methods are discussed and compared below.
Synthetic strategies for labeling IgG with Nanogold. (Top) Labeling at a hinge thiol with Monomaleimido Nanogold gives the greatest site-specificity; however, labeling at an Fc sugar residue (middle) ensures that labeling is remote from the antigen binding regions, while using Mono-Sulfo-NHS-Nanogold to label at the N-terminal position or at a lysine residue (bottom) can provide a more straightforward synthesis, and works on most types of IgG under similar conditions.
Hinge thiol labeling with Monomaleimido Nanogold®
This method provides the greatest degree of control over labeling. Use it for:
The thiol group that is used for labeling is obtained by the reduction of a hinge disulfide, which is usually conducted using a reducing agent such as dithiothreitol (DTT), mercaptoethylamine hydrochloride (MEA) or mercaptoethanol. The susceptibility of the hinge thiols to reduction varies between different IgG types and species, but the optimum concentration for reducing hinge disulfides while leaving disulfides elsewhere intact is usually between 10 mM and 50 mM: reduction is usually carried out for 60 to 90 minutes at slightly elevated temperature (37°C). The antibody is then separated from the reducing agent using gel filtration over a desalting column such as GH25 desalting gel (contact Millipore): this is required since the thiol-based reducing agent will react with maleimides. The reduced IgG is then mixed with Monomaleimido Nanogold at pH 6.5 at room temperature, agitated gently for one hour, then incubated overnight at 4°C to ensure that the reaction is complete. The labeled IgG is then separated from excess Nanogold by gel filtration using a Superose-12 or similar column.
- High resolution electron microscopic labeling.
- Antigen quantitation and other quantitative labeling applications.
- Where you have sufficient antibody to repeat the labeling reaction if labeling is low first time.
Although we have not extensively tested this alternative, the labeling procedure may be simplified by using a non-thiol based reducing agent, such as sodium borohydride (NaBH4) or sodium cyanoborohydride (NaBH3CN), which do not react with maleimide. This removes the requirement to separate the reducing agent; however, allowing it to quench before adding the Monomaleimido Nanogold is recommended, as these may still possess some reactivity towards components of the Nanogold label.
- Most site-specific labeling.
- Conjugates labeled at the hinge region give highest electron microscope labeling resolution.
- Attachment site is positioned away from both the antigen combining and Fc regions, allowing both to participate in binding reactions (for example, binding a tertiary probe).
- Moderately complex labeling procedure: requires gel filtration to separate thiol-based reducing agents.
- Hinge thiols in IgGs from different species vary in their susceptibility to reduction. Some trial and error may be necessary to identify the optimum conditions for hinge thiol reduction.
Fc labeling using periodate oxidation and Monoamino Nanogold®
Labeling at the Fc region is somewhat more challenging than hinge thiol labeling, because the Fc region has less well-defined functional groups. However, it does have one unique group that is present in the Fc region of antibodies: carbohydrates, or sugar residues. You can use mild oxidation with sodium periodate to convert these to reactive aldehydes; the aldehydes may then be labeled with Monoamino Nanogold®, followed by reduction with cyanoborohydride to reduce the resulting Schiff base to a secondary amine. This is the same procedure that we recommended in our Application Note on RNA labeling. You should consider this approach when:
- You have a limited amount of antibody, require labeling away from the antigen binding region, but do not know the optimum conditions for hinge disulfide reduction.
- You wish to leave the hinge region for subsequent reaction.
Alternatively, it may be possible to convert the aldehydes to carboxylic acids, and this may make labeling easier: the carboxylic acid may be converted to a reactive ester using either 1-Ethyl-3-[3-dimethylaminopropyl] carbodiimide Hydrochloride (EDC) with Sulfo-N-hydroxysuccinimide or N,N-carbonyldiimidazole in DMF: the activated ester is then reacted with Monoamino Nanogold. However, caution should be exercised that the activated esters do not react with amino- groups elsewhere in the IgG molecule.
You can also cross-link to aldehydes directly using a carbonyl-reactive hydrazido- maleimide cross-linkers; a variety are available. Succinimidyl 6-(3-[2-pyridyldithio]-propionamido) hydrazide (SPDP hydrazide) introduces a disulfide, which is readily reduced to a sulfhydryl and labeled with Monomaleimido Nanogold. Others introduce maleimides, which may then be reacted with mercaptoethylamine hydrochloride to convert them to amines, and labeled with Mono-Sulfo-NHS-Nanogold. You can find suitable cross-linkers from the list of heterobifunctional cross-linkers from Molecular Biosciences, or the cross-linker selection guide from Pierce.
- Nanogold label is located well away from the antigen combining region, so maximum immunoreactivity is preserved.
- Hinge thiol is preserved for maximum structural integrity.
- Procedure is more straightforward than hinge thiol labeling, since gel filtration is not required before Nanogold addition.
- A variety of cross-linking options are available.
- Labeling results may be variable.
- Because the gold is positioned away from the binding region, resolution in the electron microscope is lower than it would be with hinge thiol labeling.
- Oxidation and reduction reactions can produce multiple labeling sites, making labeling more difficult to control than hinge thiol labeling.
N-terminal or lysine residue amine labeling using Mono-Sulfo-NHS-Nanogold®
The simplest method for IgG labeling, but also the least specific, is to react directly with Mono-Sulfo-NHS-Nanogold®. This reagent will label any accessible amine, whether the N-terminal amine, or a lysine residue or other amino-functionalized amino acid residue or modification. Because IgG molecules usually contain multiple amine sites, this approach engenders a high level of confidence that at least one will be labeled with little or no optimization, and this approach is useful if you have a very small amount of antibody to work with or need a fast, preliminary result. Because no modification is needed prior to conjugation, native antibody structure is preserved best with this method, and it is therefore also recommended if other approaches result in changes to the immunoreactivity or structure of the IgG, or a loss of stability after conjugation. This approach is useful if:
- You have very little antibody or a very limited budget, and cannot afford to repeat the labeling reaction.
- You need high ratios of gold to antibody: because most IgG molecules contain multiple amines, the likelihood of conjugating more than one Nanogold per antibody is higher than with hinge thiol labeling.
- You have limited preparation time or your access to chromatography facilities is restricted.
- If other methods result in loss of stability or antibody reactivity, or structure or MW changes.
The IgG is dissolved in a non-amine-containing buffer, such as phosphate-buffered saline or Tris-NaOH, at pH 7.5 to 8.2. Higher pH produces faster labeling, and may improve labeling efficiency, although it will also accelerate the competing hydrolysis of the NHS ester. The Mono-Sulfo-NHS-Nanogold is then reconstituted in water and mixed with the protein, agitated gently for 45 minutes to one hour, then incubated overnight at 4°C to ensure completion of the reaction. Next day, the labeled IgG is separated from excess Nanogold by gel filtration using a Superose-12 or similar column.
- Simplest procedure for IgG labeling, and requires least preparation and equipment use.
- Reaction most likely to work first time.
- High ratios of Nanogold to IgG are possible.
- Native IgG structure is best preserved, and may result in higher conjugate stability.
- Because labeling can occur in any region of the IgG, this approach provides least control over the conjugation site and ratio of Nanogold to IgG.
- Labeling may occur close to antigen binding region, possibly compromising immunoreactivity.
- Conjugates labeled in this manner may provide lower resolution than others in macromolecular electron microscopic localizations.
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Gold enhancement is an alternative to silver enhancement, developed by Nanoprobes. With gold enhancement, gold nanoparticles, gold - instead of silver - is deposited onto colloidal gold or gold cluster labels. This catalytic enlargement and enhancement process produces enlarged particles for electron microscopic observation and dark staining for light microscopy and blotting.
Gold enhancement has important advantages over silver enhancement for several applications:
- Cleaner signals with lower background for light microscopy and blotting.
- Osmium etch resistance for EM: may safely be used before any strength osmium tetroxide.
- Compatible with physiological buffers: does not precipitate with halides.
- Compatible with metal substrates for cell culture or biomaterials.
- Less pH sensitive than silver enhancement: use in a wider pH range.
- Better SEM Visualization: much stronger backscatter signal than silver.
- Near neutral pH for best ultrastructural preservation, and low viscosity for ease of use.
Taguchi and group used the osmium resistance and enhanced SEM signal of gold-enhanced FluoroNanogold to their advantage in their recent paper describing the development of a novel technique, Instant with DTT, EDT, And Low temperature (IDEAL)-labeling, for rapid and specific FlAsH-labeling of tetracysteine-tagged cell surface proteins using new fluorescent biarsenical (FlAsH) derivatives for labeling cellular and extracellular proteins, published recently in Molecular Biology of the Cell. The authors used prion protein (PrP) expressed and amyloid precursor protein (APP), expressed in N2a cells with a fused tetracysteine motif, as models; labeling was confirmed by using a biotinylated tetracysteine-binding probe (Bio-FlAsH) which was the detected with Alexa Fluor® 594-streptavidin-FluoroNanogold. Cells were grown on precleaned coverslip-bottom dishes. After Bio-FlAsH labeling, they were rinsed once with Hanks balanced Salt solution and chilled to 4°C in PBS, then labeled for 20 minutes at 4°C with a 1 : 20 dilution of Alexa Fluor 594-streptavidin-FluoroNanogold in PBS + 2% bovine serum albumin (BSA) + 0.1% BSA-c (Electron Microscopy Sciences, Hatfield, PA). After multiple washes with ice-cold PBS + 0.1% BSA-c, one of which included 10 mM d-biotin, the cells were fixed with 4% paraformaldehyde / 5% sucrose in PBS for 20 minutes, then rinsed with PBS before imaging.
Cells were then analyzed by scanning electron microscopy. Cells grown on clean silicon chips and labeled with Bio-FlAsH were fixed in Karnovskys fixative at room temperature, then developed with GoldEnhance EM for 8 minutes. Samples were then postfixed with an OTO method with 1% osmium tetroxide in cacodylate buffer using microwave-assisted processing, treated with saturated thiocarbohydrazide, washed twice with water, dehydrated with ethanol, and critical-point dried through carbon dioxide. The silicon chips were then mounted on aluminum stubs, lightly coated with chromium using an ion beam sputterer and imaged at 5 kV on a Hitachi S4500 field emission scanning electron microscope in mixed mode to simultaneously detect backscattered and secondary electrons. C3 generation and N-terminal truncation of PrPres were inhibited by the anti-prion compound E64, a cysteine protease inhibitor: E64 did not inhibit the synthesis of new PrPres, providing insight into the mechanism by which E64 reduces steady-state PrPres levels in prion-infected cells. Using the new probes, IDEAL labeling could be used to extend the use of biarsenical derivatives to extracellular proteins and beyond microscopic imaging.
- Taguchi, Y.; Shi, Z. D.; Ruddy, B.; Dorward, D. W.; Greene, L., and Baron, G. S.: Specific biarsenical labeling of cell surface proteins allows fluorescent- and biotin-tagging of amyloid precursor protein and prion proteins. Mol. Biol. Cell, 20, 233-244 (2009).
Enhancement of Nanogold® by GoldEnhance: mechanism. Final particle size is controlled by enhancement time; particles may be enlarged to sizes between 3 nm (1-2 minutes) and 50 nm or larger (10 minutes and longer).
GoldiBlot combines simple gold labeling of His-tagged proteins with a rapid gold-based autometallographic amplification process, in which metal is selectively deposited onto the bound gold particles. The principle is shown below, together with results obtained for staining His-tagged proteins in a Western blot. Unlike the metal enhancement processes used for organic chromogens, this produces a clean, clear signal without the need for additional reagents or steps.
Left: Western blot detection of His-tagged proteins using GoldiBlot HIS Protein Detection kit. Lane M: All Blue protein ladder. Lanes 1-5: His-tagged ATF-1 loaded at 2.5 50 ng (1) 50 ng, (2) 25 ng, (3) 10 ng, (4) 5 ng and (5) 2.5 ng. (6) 100 ng His-tagged YY1. (7) 100 ng His-tagged Src. (8) 50 ng His-tagged Src and bacterial extract with 2,500 ng total E. Coli Protein. (9) bacterial extract with 2,500 ng total E. Coli Protein. Right: How GoldiBlot works: Ni-NTA-Gold binds to His-tagged proteins, and the gold particles are then subjected to autometallographic amplification to render them visible.
Features and advantages include:
- Detect His-tagged proteins in an hour.
- Direct visualization of His-tagged proteins in magenta colored bands. No film, autoradiography or phosphorimager are required.
- More stable than antibodies.
- Low nanogram-level sensitivity with low background.
- No antibodies involved.
gold enhancement also yields much improved results over silver enhancement in conventional immunoblots, providing substantially lower background while maintaining a similar level of sensitivity to silver enhancement. If you have been using Nanogold®: for detection on nitrocellulose membrane blots (immunodot blots or Westerns), we have developed an optimized detection procedure that maintains the already very high sensitivity, but combines it with a greatly reduced background and enhanced signal clarity. For best results, we recommend using a procedure that incorporates the following features:
- Identify His-tagged proteins rapidly and confidently in cell lysates and extracts.
- Faster, simpler western blots.
- Confirm the expression of his-tagged reporter proteins in transfected cells.
- Incorporate 0.1% Tween-20 (detergent) in the buffers used for blocking, antibody incubation, and washing. This will dramatically reduce background binding.
- Include 1% nonfat dried milk (you can use the material sold in supermarkets and food stores) as an additive in the incubation buffer (the buffer in which the Nanogold is dissolved and applied to the blot) and 5% nonfat dried milk in the blocking buffer used to block the membrane before application of antibodies.
REAGENTS AND EQUIPMENT:
- Phosphate buffered saline (PBS): 20 mM sodium phosphate buffer pH 7.4 and 150 mM NaCl.
- Specific antigen (target protein or other biomolecule).
- Nitrocellulose (NC) membrane 0.2 µm pore size.
- Blotting Paper to wick membrane dry.
- Orbital Shaker
- Washing buffer (TBS-Tween 20): 20 mM Tris pH 7.6, with 150 mM NaCl and 0.1 % Tween-20.
- Nonfat dried milk (Carnation)
- GoldEnhance EM (Nanoprobes Product No. 2113).
- Specific Nanogold antibody conjugate.
- Prepare antigen solutions with a series of dilutions (0.01mg/mL, 0.001mg/mL, 0.0005mg/mL, 0.0001 mg/mL, 0.00005 mg/mL, 0.00001 mg/mL and 0.000005 mg/mL) using PBS, pH7.4.
- Pipette 1 µL of above solutions to a dry nitrocellulose membrane. Prepare two duplicates as a negative control: (1)Negative control 1: No antigen, No antibody; and (2) Negative control 2: No antigen, but with NG-conjugate incubation.
- Air-dry for 30 minutes
- Immerse membranes in 8 mL of TBS-Tween 20 for 5 minutes.
- Block membranes in 8 mL of TBS-Tween 20 containing 5 % nonfat dried milk for 30 minutes at room temperature.
Binding of Nanogold antibody conjugate:
- Dilute Nanogold antibody conjugate in TBS-Tween 20 containing 1% nonfat dried milk to 4 µg/mL (1:20 Dilution: 300 µL conjugate + 5.30 ml TBS-gelatin containing 1% nonfat dried milk).
- Incubate the membranes in 8 mL of diluted conjugate solution for 30 minutes at room temperature.
- Incubate the control membrane in 8 mL of TBS-Tween 20 containing 1% nonfat dried milk for 30 minutes at room temperature.
- Wash membranes three times for 3 minutes each in 8 mL of TBS-Tween 20. Wash membranes thoroughly in 8 mL of deionized water (4 x 3 minutes). Make sure strips are washed separately according to what they are incubated in (strips incubated in one lot of a conjugate are washed in a separate dish from strips that are incubated in TBS-Tween 20 with 1% nonfat dried milk without conjugate, strips incubated in different lots are washed separately).
- Perform Gold Enhancement according to instructions (mix solutions A and B, wait 5 minutes, then add C and D).
- Record the number of observed spots and time when the spots appear. Record the time when background appears on the control membrane.
- After 15 minutes, the enhancement solution is removed. Rinse membranes with water (3 x 3 minutes) and air-dry for storage.
If you need to use silver enhancement rather than gold enhancement, Tween 20 and nonfat dried milk should also improve the performance of Nanogold conjugates used with silver enhancement. Examples of blot detection with Nanogold using both gold and silver enhancement on blots are shown below, in comparison with detection using conventional colloidal gold.
Blot test with Nanogold and colloidal gold conjugates. (a) dot blot image of Nanogold-Fab' Goat anti-Rabbit IgG, developed with GoldEnhance EM gold enhancement reagent, using the Schleicher & Schuell (recently acquired by Whatman) MINIFOLD-1 Dot Blot System which applies the target precisely over a circle with a diameter of 5 mm. The 5 mm circle is big enough for a quantitative analysis by a densitometer. With the target spreading over 5mm circle, we can detect as low as 0.01 ng antigen. While we use a pipette to apply 1 L of target over a circle of 1-2 mm, we can detect as little as 0.005 ng by the same NG conjugates. (b) Nanogold anti-mouse Fab' blotted against mouse IgG, developed with LI Silver (Nanoprobes), showing sensitivity enhancement with smaller spot size. Target was applied using a 1 µL microcapillary tube. This immunodot blot shows 0.1 pg sensitivity (arrow). (c) Colloidal gold blot using a 15 nm colloidal gold conjugate to detect serial dilutions of an IgG target, without silver or gold enhancement. The range of color densities should give a good basis for comparing your results - i.e. if your spot is darker than our 50 ng spot your conjugate is working very well, but if it is much fainter, it is questionable.
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Negative stains, such as our NanoVan (methylamine vanadate) and Nano-W (methylamine tungstate), are particularly useful for studies of virus and protein ultrastructure, and are useful in identifying aberrant protein crystallization in conditions such as Alzheimer's Disease. NanoVan is an intermediate density stain which is used to define the edges of particulate specimens in suspension for electron microscopic observation. It has a highly amorphous structure and fine grain, which provides maximum clarity and least interference in the observation of ultrastructural features at very high resolution. NanoVan is ideal for use with smaller gold labels such as Nanogold® because the stain is less electron-dense than other negative stains such as uranyl acetate or lead citrate, so sufficient contrast is produced between the gold particle, their environment, and the negative stain to differentiate them.
Left:Schematic showing how negative stains work. Right: high-resolution electron micrographs obtained using a scanning transmission electron microscope. (a) Tobacco Mosaic Virus (TMV) negatively stained with 2 % uranyl acetate; (b) TMV stained with 1 % methylamine vanadate (NanoVan); both samples imaged with a dose of 104 eI/nm2. Original full width 128 nm for each image. (c) Side view of groEL (large arrow) labeled with 1.4 nm gold cluster (Nanogold, small arrow) imaged in methylamine vanadate. Note clear visibility of subunit structure and gold cluster. Full width 128 nm. Specimen kindly provided by A. Horwich, Yale University.
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.
Tagliabracci and group added to the applications of NanoVan in their recent study of the role of abnormal glycogen phosphorylation in Lafora disease, which appeared recently in the Journal of Biological Chemistry. Lafora disease is a progressive myoclonus epilepsy with onset in the teenage years, followed by neurodegeneration and death within 10 years. A characteristic symptom is the widespread formation of poorly branched, insoluble glycogen-like polymers (polyglucosan), known as Lafora bodies, which accumulate in neurons, muscle, liver, and other tissues. About half of Lafora disease cases present with mutations in the EPM2A gene. This encodes laforin, a protein phosphatase that releases the small amount of covalent phosphate normally present in glycogen, and this led the authors to hypothesize that abnormal phosphorylation of glycogens is a factor in the disease.
In studies of Epm2a-/- mice, which lack laforin, a progressive change in the properties and structure of glycogen was observed. After three months, glycogen metabolism remained essentially normal, but phosphorylation of glycogen had increased 4-fold and the polysaccharide had begun to demonstrate altered physical properties. At 9 months, the glycogen had overaccumulated by 3-fold and become somewhat more phosphorylated. It was also poorly branched, and insoluble in water. Negative stain electron microscopy was used to study its morphology. Glycogen was extracted from WT or Epm2a-/- mouse skeletal muscle or liver by boiling the tissue was boiled in 30% KOH and filtering to remove floating fat. The glycogen was precipitated with ethanol and redissolved in water. Ten volumes of 4:1 methanol/chloroform was added, and the solution was mixed and heated at 80°C for 5 minutes. Glycogen was recovered, ethanol precipitated, and redissolved in 10% TCA. The solution was centrifuged, and the glycogen was recovered from the supernatant by ethanol precipitation, dialyzed, and re-precipitated again with ethanol to remove lipids. Purified glycogen (1525 µg) was spotted onto a Formvar-coated grid and allowed to settle for 3060 seconds. A drop of NanoVan was then added, and wicked off after 30 seconds. Specimens were viewed with a Technia G12 Biotwin transmission electron microscope equipped with an AMT CCD camera at 80 kEV and 150,000 X magnification. Particle diameters were measured, and a histogram was constructed depicting the size distribution of the particles for different age groups and genotypes.
Glycogen from wild-type mice usually appears as rosettes with a characteristic granular structure. However, negative stain electron microscopy revealed a strikingly abnormal morphology in extracts from 9 and 12-month Epm2a-/- mice, which paralleled the formation of Lafora bodies. The glycogen molecules showed a tendency to aggregate, and glycogen could be pelleted by low speed centrifugation of tissue extracts. The resulting particles were larger, with a more distinct boundary, less granularity, and higher density than the wild type. This aggregation requires the phosphorylation of glycogen. Treatment with laforin partially resulted in reduced particle size and incomplete restoration of granularity, indicating that excessive phopshorylation is a factor in the altered morphology. The aggregrated glycogen sequesters glycogen synthase, but not other glycogen metabolizing enzymes. The authors concluded that laforin functions to suppress excessive glycogen phosphorylation, and is essential for the formation of normally structured glycogen.
- Tagliabracci, V. S.; Girard, J. M.; Segvich, D.; Meyer, C.; Turnbull, J.; Zhao, X.; Minassian, B. A.; Depaoli-Roach, A. A., and Roach, P. J.: Abnormal metabolism of glycogen phosphate as a cause for Lafora disease. J. Biol. Chem., 283, 33816-33825 (2008).
Reference for sample preparation:
- Tagliabracci V. S.; Turnbull J.; Wang W.; Girard J. M.; Zhao X.; Skurat A. V.; Delgado-Escueta A. V.; Minassian B. A.; Depaoli-Roach A. A., and Roach P. J.: Laforin is a glycogen phosphatase, deficiency of which leads to elevated phosphorylation of glycogen in vivo. Proc. Natl. Acad. Sci. USA, 104, 19262-19626 (2007).
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Beat the Price Increase - Order Before February 15
Unfortunately, our costs have increased over the last few months, and as a result, we will need to raise our prices by approximately 3% on all our products. The increase will be effective February 15, so order before this date to avoid the increase and pay 2008 prices.
We are currently updating our product instructions to incorporate improved procedures, remove unnecessary protocols, and add details that ensure that you know how to obtain the best performance. We have recently updated the instructions for our silver enhancers and gold enhancement reagents, as well as for AuroVist and Fluorescein FluoroNanogold. Look for updates to other product instructions in the near future.
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Other Recent Publications
SERS, or surface-enhanced Raman scattering, is another application of metal nanoparticles, primarily colloidal silver, that provides a method for the analysis of bonding within molecules adsorbed to the surface of the particles. The concentration of target molecule at the silver surface enhances the Raman signal, and because it changes the symmetry of the molecule, can also enhance or modify some of the vibrational modes of the molecule to provide new data on its electronic structure and bonding. In the current edition of Nano Letters, Weiyang Li and co-workers explore the use of dimers of silver nanoparticles to generate 'hot spots' with enhanced SERS activity. The authors used a simple, one-pot method to generate dimers of silver nanospheres in one step: synthesis is based upon the polyol process, in which ethylene glycol (EG) serves as a solvent and a precursor to the reducing agent. A small amount of sodium chloride was introduced into the reaction solution, resulting in the formation of single-crystal silver nanoparticles through oxidative etching, and at the same time inducing dimerization due to a change to colloidal stability (in a similar manner to the aggregation of colloidal gold promoted by sodium chloride). Dimer formation was maximcized by control of colloidal stability and oxidative etching, by optimizing the amount of chloride added to the polyol synthesis. The dimers consisted of single-crystal silver nanospheres ~30 nm in diameter, separated by a gap of 1.8 nm wide. The dimers provide a well-defined system for studying the hot spot phenomenon (hot spot: the gap region of a pair of strongly coupled silver or gold nanoparticles), an extremely important but poorly understood subject in surface-enhanced Raman scattering (SERS). Because of the relatively small size of the silver nanospheres, only those molecules trapped in the hot spot region are expected to contribute to the detected SERS signals. The authors used silica coating to trap the dimers for electron microscopic observation: by correlating SERS measurements with SEM imaging, they found that the SERS enhancement factor within the hot spot region of such a dimer was on the order of 2 x 107.
Chad Mirkin and group continue to contribute understanding of processes that occur in gold nanoparticle constructs, and they present two papers in January's Nano Letters that yield novel insights into the properties of DNA-gold conjugates. In the first, they report on the hybridization interactions of gold-conjugated oligonucleotides. 60 nm gold nanoparticles and 140 nm edge length triangular gold nanoprisms were functionalized with a series of 24-mer propylthiol-modified oligonucleotides. Melting temperature experiments suggested that DNA strands chemically immobilized on gold nanoparticle surfaces can engage in two types of hybridization: one that involves complementary strands and normal base pairing interactions, and also a second, assigned as a "slipping" interaction, which can additionally stabilize the aggregate structures through non-Watson-Crick type base pairing or interactions less complementary than the primary interaction. Comparison of the results from nanoparticles and prisms indicated that particle curvature is a major factor that contributes to the formation of these slipping interactions; flat gold triangular nanoprism conjugates of the same sequence do not support them. These slipping interactions were found to significantly stabilize nanoparticle aggregate structures, leading to large increases in Tms and effective association constants compared with free DNA and particles lacking the appropriate sequence to maximize their contribution.
- Li, W.; Camargo, P. H. C.; Lu, X., and Xia, Y.: Dimers of Silver Nanospheres: Facile Synthesis and Their Use as Hot Spots for Surface-Enhanced Raman Scattering. Nano Lett., 9, 485-490 (2009).
- Hill, H. D.; Hurst, S. J., and Mirkin C. A.: Curvature-Induced Base Pair "Slipping" Effects in DNA-Nanoparticle Hybridization. Nano Lett., 9, 317-321 (2009).
In the second paper, the group investigate the mechanisms by which DNA-gold conjugates resist enzymatic degradation. Polyvalent oligonucleotide gold nanoparticle conjugates have unique fundamental properties, including distance-dependent plasmon coupling, enhanced binding affinity, and the ability to enter cells and resist enzymatic degradation. This stability in the presence of enzymes is a key consideration for therapeutic uses, and therefore understanding of how this arises is an important factor in the development of gold nanoparticle-oligonucleotide therapeutics. The authors used 13 nm gold particles conjugated with a monolayer of oligonucleotides comprising a 20-base DNA sequence, a 10-base DNA linker, and propylthiol anchor, hybridized with a fluorescein-labeled complementary sequence; in this state, fluorescence is quenched. Susceptibility to enzymatic digestion was measured by the addition of deoxyribonuclease I (DNase I) and monitoring of the rate of fluorescence increase as the fluorescently labeled strands were released from the particle surface. The enhanced stability of polyvalent gold oligonucleotide nanoparticle conjugates was evaluated in comparison with enzyme-catalyzed hydrolysis of DNA; the results support a conclusion that the negatively charged surfaces of the nanoparticles and resultant high local salt concentrations are responsible for enhanced stability.
- Seferos D. S.; Prigodich A. E.; Giljohann D. A.; Patel P. C., and Mirkin C. A.: Polyvalent DNA nanoparticle conjugates stabilize nucleic acids. Nano Lett., 9, 308-311 (2009).
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