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Updated: November 7, 2003

N A N O P R O B E S     E - N E W S

Vol. 4, No. 11          November 7, 2003


This monthly newsletter is to keep you informed about techniques to improve your immunogold labeling, highlight interesting articles and novel metal nanoparticle applications, and answer your questions. We hope you enjoy it and find it useful.

Have questions, or issues you would like to see addressed in the next issue? Let us know by e-mailing [email protected].

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FluoroNanogold Shows 3D Organization of Pki-67

Although monoclonal antibody to Ki-67 is often used clinically to estimate tumor growth fraction, the role of the Ki-67 protein itself has remained elusive. Cheutin and co-workers have used correlative confocal microscopy, electron microscopy and electron tomography using FluoroNanogold labeling to determine its three-dimensional organization in cultured human interphase A549 cells. Confocal microscopy followed by 3D reconstruction showed that pKi-67 forms a shell around the nucleoli, and double labeling experiments revealed that pKi-67 co-localizes with perinucleolar heterochromatin. Electron microscopy confirmed this close association and demonstrated that pKi-67 is located neither in the fibrillar nor in the granular components of the nucleolus. Finally, spatial analyses by electron tomography showed that pKi-67 forms cords 250 - 300 nm in diameter, which are themselves composed of 30 - 50-nm-thick fibers. These detailed comparative in situ analyses strongly suggest the involvement of pKi-67 in the higher-order organization of perinucleolar chromatin.

After simultaneous fixing and permeabilization for 4 min in 3 % paraformaldehyde and 1 % Triton X-100 in PBS (140 mM NaCl, 6 mM Na2HPO4, 4 mM KH2PO4, pH 7.2), cells were saturated for 30 min in PBS containing 3% bovine serum albumin (BSA), 1 mM CaCl2, and 0.5 mM MgCl2, then incubated for 30 minutes with MM1 antibodies against human pKi-67 diluted 1:50 in PBS containing 1 % BSA, 1 mM CaCl2, and 0.5 mM MgCl2 and rinsed (3 X 5 min) in PBS. Biotinylated goat anti-mouse antibody was applied for 30 minutes, then revealed for 15 minutes with either streptavidinFluoroNanogold (diluted 1:20), or streptavidin-Texas Red conjugate (diluted 1:50). Staining of DNA was performed for 5 minutes at room temperature using 100 M chromomycin A3 diluted in PBS containing 150 mM MgCl2. For confocal microscopy studies, cells on coverslips were mounted in Citifluor AF1. For electron microscopy studies, cells immunolabeled with FluoroNanogold were over-fixed for 12 min with 1.6 % glutaraldehyde in PBS, rinsed in de-ionized water, and HQ silver enhancement was performed for 8 min. Cells were harvested by scraping, dehydrated in graded alcohols, and embedded in Epikotte 812.

Reference:

Cheutin, T.; O'Donohue, 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, 1411-1423 (2003).

Abstract (Medline):
http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=14566014&dopt=Abstract

This group used the same method previously to localize RNA polymerase I (RPI) in the same cell line for a study to localize transcribing rRNA genes at the ultrastructural level and described their three-dimensional organization within the nucleolus by electron tomography.

Reference:

Cheutin, T.; O'Donohue, M. F.; Beorchia, A.; Vandelaer, M.; Kaplan, H.; Defever, B.; Ploton, D., and Thiry, M.: Three-dimensional organization of active rRNA genes within the nucleolus. J. Cell. Sci., 115, 3297-3307 (2002).

Meanwhile, Lujan, Touz and co-workers used FluoroNanogold to localize Encystation-specific cysteine protease (ESCP) in the intestinal parasite Giardia lamblia. Reference:

Touz, M. C.; Lujan, H. D.; Hayes, S. F.; and Nash, T. E.: Sorting of encystation-specific cysteine protease to lysosome-like peripheral vacuoles in Giardia lamblia requires a conserved tyrosine-based motif. J. Biol. Chem., 278, 6420-6426 (2003).

Abstract (Medline):
http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=12466276&dopt=Abstract

The method is described in detail by Hayman and group: after monoclonal primary antibody incubation and PBS-BSA washes, the FluoroNanogold anti-mouse conjugate, diluted 1:30 in PBS-BSA with either 0.05 or 0.025 % saponin, was added for 1 h at room temperature. The coverslips were washed five times in PBS and stored at 4°C in postfixative (2.5% glutaraldehyde, 4 % paraformaldehyde) until used. For electron microscopy, the coverslips were washed in water and enhanced for 4 minutes in the dark with HQ Silver, then washed three times in water and once in 1 % aqueous tannic acid for 5 min, then once more in water. Next, they were reacted with a solution of reduced K4(FeCN)6 and 1% osmium tetroxide for 15 min, followed by two rinses in water, subjected to a 5-min graded alcohol dehydration series of 50, 80, 95, and 100%, infiltrated with Spurrs resin, and polymerized at 60°C.

Reference:

Hayman, J. R.; Hayes, S. F.; Amon, J.; and Nash, T. E.: Developmental expression of two spore wall proteins during maturation of the microsporidian Encephalitozoon intestinalis. Infect. Immun., 69, 7057-7066 (2001).

In addition to the fluorescein version, we now offer FluoroNanogold conjugates prepared with the brighter, more stable Alexa Fluor® 488 and 594 dyes. More information:

Alexa Fluor® is a registered trademark of Molecular Probes, Incorporated.

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Nanogold® Labeling: Multiple Conjugation, and Ionization

Some selections from our technical help files on issues raised recently:

(a) Can I link more than one molecule to Nanogold®?

Positively Charged Nanogold® and Negatively Charged Nanogold® both contain multiple functional groups (amines and carboxyls respectively) that may be activated for cross-linking. Therefore, it is feasible to link more than one entity to them. However, while this is an effective strategy for linking several copies of the same entity, we do not recommend it for successive conjugation reactions. Only one type of reactivity is incorporated into Nanogold. This makes it extremely difficult to control the reaction stoichiometry to ensure that the first conjugation yields predominantly the desired conjugate, while sufficient unreacted functionalities remain for the second reaction. In addition, if you have activated the gold, it will be very difficult to remove either gold that has failed to react in the first step, or unconjugated biological molecule, before the activated group is hydrolyzed.

In many cases, you may be able to link the three components together in a different conformation much more easily. If one is a larger molecule that has more than one reactive functional group, then this will be the one that can support multiple attachments, and you can label it sequentially at two different sites using different cross-linking chemistries. This is how FluoroNanogold is made: first Monomaleimido-Nanogold is linked to a hinge thiol, then an amine-reactive fluorescent label is conjugated to an amine elsewhere in the Fab' fragment.

For example, if you wish to link both Nanogold and an oligonucleotide to an antibody, the best strategy is to first label the IgG with Monomaleimido-Nanogold, to attach the gold selectively to the hinge region. This positions the gold away from the antigen combining region, and leaves the amino- groups elsewhere on the molecule intact for the second step. Then, once you have purified the Nanogold-IgG conjugate, activate the DNA for cross-linking to amines - for example, incorporate a 5'-amino modifier during synthesis and activate with a heterobifunctional cross-linker such as bis-(sulfo-scuccinimidyl)-suberate, or BS3. React this with the IgG. Of course, if you decide it makes more sense to position the oligo in the hinge region, you can do this the other way round - activate the oligonucleotide for thiol reactivity, couple to the hinge region of the reduced IgG, then label the conjugate with Mono-Sulfo-NHS-Nanogold (remember not to use thiolated reagents after Nanogold labeling, as these can displace or degrade the gold).

(b) Does Nanogold® or undecagold ionize?

Many of you wish to use charge-based separation methods, such as gel electrophoresis or ion exchange chromatography, to isolate gold conjugates, and are interested to know what charge the Nanogold® and undecagold reagents have.

Both Nanogold and undecagold are molecular coordination compounds. The gold core itself does not have any residual charge, but is fully stabilized and capped by coordinated ligands. The only charge that these molecules have is that of chemical groups that we incorporate into the coordinated ligands to impart reactivity or functionality. For example, Positively Charged Nanogold is protonated at intermediate to low pH values; Negatively charged Nanogold is deprotonated at mid to high pH values. However, when Monomaleimido- and Mono-Sulfo-NHS-Nanogold react as intended, they form respectively a thioether or an amide cross-link. Neither of these may be protonated or deprotonated under the conditions in which gold conjugates are stable.

This means that labeling with Monomaleimido Nanogold will not affect the charge of the conjugate biomolecule, and therefore the only change it will impart in charge-based separation procedures is that of the added mass of the gold (although other factors may affect gel electrophoresis separation). Labeling with Mono-Sulfo-NHS-Nanogold will remove one amine from the conjugate biomolecule, and its ionization behavior will be modified accordingly.

More information and help:

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NanoVan Negative Staining Reveals Prion Architecture

Negative stains are used with particulate or suspended specimens such as protein complexes: the stain fills in the gaps between features of interest to contrast their edges. Nanoprobes offers two negative stain reagents with complementary properties. NanoVan is recommended for use with Nanogold® because it is based on vanadium and therefore is less electron-dense than heavier metal-based stains such as uranyl acetate. Nano-W is based on the heavier element tungsten, and therefore gives a more dense stain. It is more suited to use with larger gold labels. These two reagents are completely miscible, and therefore may be mixed together in different proportions to generate any desired intermediate stain density. Both have near-neutral pH, and NanoVan has been found to be less susceptible to electron beam damage than uranyl acetate.

The resolution of the results obtained with negative stains depends upon the lack of crystallinity in the stain. NanoVan is highly amorphous, and its advantages for high-resolution scanning transmission electron microscopy were recently demonstrated by Baxa and group, who used it as part of a study that included both cryoelectron microscopy and the use of STEM to calculate masses of Ure2p prion filaments. Protease digestion of 25 nm diameter filaments of native Ure2 protein, a regulator of nitrogen catabolism in yeast, and a fusion protein yielded 4 nm filaments: mass spectrometry and STEM mass measurements indicated that these consisted of prion domains. A unifying model was inferred whereby subunits in Ure2p filaments are connected by interactions between their prion domains, which form a 4-nm amyloid filament backbone surrounded by the corresponding C-terminal moieties.

Reference:

Baxa, U.; Taylor, KL.; Wall, JS.; Simon, M. N.; Cheng, N.; Wickner, R. B., and Steven, A. C.: Architecture of Ure2p Prion Filaments: THE N-TERMINAL DOMAINS FORM A CENTRAL CORE FIBER. J. Biol. Chem., 278, 43717-43727 (2003).

Abstract (Medline):
http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=12917441&dopt=Abstract

More information:

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Nanogold® and the Chloroplast Outer Envelope Translocon

Enrico Schleiff and co-workers provided another demonstration of the utility of Nanogold® labeling reagents for the high-resolution labeling of specific sites within protein complexes in order to shed light on their structure and function in a recent paper in the Journal of Cell Biology. The protein translocon of the outer envelope of chloroplasts (Toc) consists of the core subunits Toc159, Toc75, and Toc34. The core complex was purified: it has an apparent molecular mass of 500 kD. Radioactive labeling and PAGE yielded a molecular stoichiometry of 1:4:45 between Toc159, Toc75, and Toc34. The isolated translocon recognizes both transit sequences and precursor proteins in a GTP-dependent manner, suggesting its functional integrity, as shown by specific binding of a Nanogold-labeled transit peptide. Two-dimensional structural analysis by negative stain molecular TEM and image analysis revealed roughly circular particles consistent with the formation of a stable core complex. The particles show a diameter of 130 Ä with a solid ring with a less dense interior structure. A three-dimensional map obtained by random conical tilt reconstruction of electron micrographs suggests that a "finger"-like central region separates four curved translocation channels within one complex.

For the Nanogold labeling reaction, 1.6 mM of the transit peptide B1, containing a COOH-terminal cysteine was incubated for 60 min at 25°C with 10 mM dithiothreitol in 0.1 M sodium phosphate, 5 mM EDTA, pH 6.0. Dithiothreitol was separated from the peptide by G25 Sephadex spin chromatography in 20 mM sodium phosphate, 150 mM NaCl, and 1 mM EDTA, pH 6.5, as running buffer. 6 nmol Monomaleimido Nanogold was dissolved in 20 microliters of dimethyl-sulfoxide and diluted 10 times into water followed by addition of 500 microliters of the reduced peptide. After incubation for 18 h at 4°C, unlabeled peptides and Nanogold were removed by passing the solution through thiol-activated sepharose. Of the flow-through, 2 microliters was added to a solution containing pelleted Toc complex, 0.5 mM GTP, and 0.5 mM MgCl2. The reaction mixture was kept 25 min on ice and subsequently used for transmission electron microscopy. Instead of GTP and MgCl2, EDTA in a final concentration of 5 mM was used as a control in the labeling reaction.

Reference:

Schleiff, E.; Soll, J.; Kuchler, M.; Kuhlbrandt, W., and Harrer, R.: Characterization of the translocon of the outer envelope of chloroplasts. J. Cell. Biol., 160, 541-551 (2003).

Abstract (Medline):
http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=12591914&dopt=Abstract

More information:

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Nanoprobes at the Association for Molecular Pathology Meeting

Our collaborators at the Cleveland Clinic Foundation will be presenting new results in the development of in situ hybridization detection methods and reagents at the upcoming Association for Molecular Pathology (AMP) meeting in Orlando, FL, November 20 - 23. Look for their presentation in Poster Session II on Saturday, November 22.

More information:

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Other Recent Publications

Zhang and co-workers have described the nonlinear optical properties of gold nanoparticles coated with goat-anti-human IgG, studied by hyper-Rayleigh scattering. The HRS signals of gold nanoparticles with IgG were larger than those of bare gold nanoparticles, and also that the HRS signals from the IgG-coated gold nanoparticles could be greatly increased when the antigen was added, due to gold nanoparticle aggregation. A measurable signal was produced with 10 microgram/mL antigen added, while the colorimetric method using UV absorption detection required 100 microgram/mL of added antigen. This suggests that HRS measurement of immunogold nanoparticles could become an immunoassay method for low levels of antigen in aqueous samples.

Reference:

Zhang, C. X, Zhang, Y, Wang, X, Tang, Z. M, and Lu, Z. H.: Hyper-Rayleigh scattering of protein-modified gold nanoparticles. Anal. Biochem., 320, 136-140 (2003).

Abstract (Medline):
http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=12895477&dopt=Abstract

An alternative method for the preparation of metal nanoparticles was advanced recently. Biffis and Speroto formed 2-3 nm platinum and palladium nanoparticles with high stability and catalytic activity inside N,N-dimethylamino-ethyl methacrylate (D) or 4-vinylpyridine (V) / ethylene dimethacrylate(EDMA) / N,N-dimethylacrylamide (DMAA) microgels; the particles are stabilized by interaction with the gel.

Reference:

Biffis, A., and Sperotto, E.: Microgel-Stabilized Metal Nanoclusters: Improved Solid-State Stability and Catalytic Activity in Suzuki Couplings. Langmuir, 19, 9548-9550 (2003).

Article information (courtesy of the American Chemical Society):
http://dx.doi.org/10.1021/la0350709

The current issue of Nature Biotechnology contains a special section on optical imaging, and includes several review papers describing recently developed techniques and imaging modes.

Table of Contents (courtesy of Nature Biotechnology):
http://www.nature.com/cgi-taf/dynapage.taf?file=/nbt/journal/v21/n11/index.html

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