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Updated: April 5, 2005

N A N O P R O B E S     E - N E W S

Vol. 6, No. 4          April 5, 2005

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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FluoroNanogold: Check Labeling before EM

Combined fluorescent and gold probes are a unique technology available exclusively from Nanoprobes, Incorporated. As described previously, they are highly effective for correlative fluorescent and gold labeling, as shown below. The FluoroNanogold product line now includes probes containing the 1.4 nm Nanogold® label and either fluorescein, Alexa Fluor®* 488 or Alexa Fluor®* 594 covalently linked to Fab' fragments or streptavidin.

[Correlative Alexa Fluor®* 594 FluoroNanogold Labeling (67k)]

Correlative fluorescence and electron microscopic labeling. Localization of caveolin-1a in ultrathin cryosection of human placenta using a new FNG; caveolin 1 alpha is primarily located to caveolae in placental endothelial cells. One-to-one correspondence is found between fluorescent spots and caveola labeled with gold particles (right). Ultrathin cryosections collected on formvar film-coated nickel EM grids were incubated with chicken anti-human caveolin-1a IgY for 30 min 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).

However, FluoroNanogold also provides a simple way of checking labeling in specimens - so you can be sure that labeling has been successful before beginning the lengthy process of embedding, sectioning and processing for electron microscopy. Braun and McBride used Alexa Fluor 488® FluoroNanogold-Fab' fragments in this manner for their recent identification of GldJ, a lipoprotein required for gliding motility in the bacterium Flavobacterium johnsoniae, which glides rapidly over surfaces.

gldJ mutants formed nonspreading colonies, and individual cells were completely nonmotile. The gldJ mutants were deficient in chitin utilization, and resistant to bacteriophages that infect wild-type cells. GldJ was cloned from a cosmid library of wild-type F. johnsoniae DNA in pCP26; cosmids were transferred into the nonmotile mutant UW102-48 by conjugation, and complemented (spreading) colonies isolated. Labeling studies with [3H]palmitate indicated that GldJ is a lipoprotein. Mutations in gldA, gldB, gldD, gldF, gldG, gldH, or gldI resulted in normal levels of gldJ transcript but decreased levels of GldJ protein, and expression of truncated GldJ protein in wild-type cells produced a severe motility defect.

The availability of a specific antiserum to GldJ protein allowed immunolocalization of GldJ in cells of F. johnsoniae. Addition of antiserum to cells did not disrupt gliding, and intact cells did not absorb significant amounts of anti-GldJ antibodies as detected by immunofluorescence microscopy, suggesting that most of the GldJ was not exposed on the cell surface. Analysis of fixed, permeabilized cells allowed detection of GldJ by immunofluorescence and immunoelectron microscopy. For immunofluorescence labeling, cells were pelleted by centrifugation, suspended in 10 mM Tris (pH 7.5), and spotted onto poly-L-lysine coated microscope slides. After incubation for 5 min at 25°C, formaldehyde was added to a 1% final concentration, and the cells fixed for 15 minutes at 25°C, then permeabilized in 100 microliters of 25 mM Tris (pH 7.5) with 5 mM EDTA and 2% Triton X-100 (2 x 30 minutes, 22°C). After washing in 25 mM sodium phosphate (pH 7.5) with 100 mM NaCl (phosphate-buffered saline, PBS) and blocking with PBS containing 1% bovine serum albumin (PBS-BSA) (30 minutes at 22°C), cells were exposed to affinity-purified anti-GldJ polyclonal antiserum (1:200 dilution) in PBS-BSA at 4&176;C for 16 hours, then washed three times in PBS and incubated with Alexa Fluor 488 anti-rabbit secondary antibody in PBS-BSA for 2 h at 22°C. For transmission electron microscopy, cells were settled onto Formvar- and polylysine-coated 400-mesh Ni grids before fixation; preparation and labeling were conducted as for immunofluorescent labeling except that washes were conducted with 1.5 ml volumes, and antibody incubations used 20 microliter volumes as droplets on Parafilm. Alexa Fluor 488®* FluoroNanogold-Fab' anti-rabbit IgG was used as the secondary antibody probe. Gold particles were then enlarged by silver enhancement (HQ Silver, 4 minutes, at 22&176;C in the dark). Samples were examined using a Hitachi H-600 transmission electron microscope at 75 kV.

In both cases GldJ was organized in discrete bands; these appeared to form a helical structure in a significant fraction of cells observed, implying that at least part of the machinery involved in cell movement is arranged in a helical array within the cell envelope.


Braun, T. F., and McBride, M. J.: Flavobacterium johnsoniae GldJ Is a Lipoprotein That Is Required for Gliding Motility. J. Bacteriol., 187, 2628-2637 (2005).

The structure of Alexa Fluor®* FluoroNanogold-Fab' and streptavidin probes, and immunofluorescence labeling with the Fab' probe, is shown below.

[Correlative Alexa Fluor®* 594 FluoroNanogold Labeling (58k)]

Structures of (a) Alexa Fluor®* 488 and Nanogold® - Fab' conjugate, and (b) Alexa Fluor®* 488 and Nanogold® - Streptavidin. (c) 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, a Mouse anti-Human secondary antibody, and combined Alexa Fluor®* 488 and Nanogold® - 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).

Since immunofluorescent and immunogold labeling sometimes work best under different conditions, combining them sometimes requires a degree of compromise between the optimum conditions for each label, and methods for ensuring that background is minimized and sensitivity optimized are at a premium. These suggestions may be helpful in achieving the best performance:

  • For increased fluorescence, use the Alexa Fluor®* conjugates rather than the fluorescein ones. The Alexa Fluor®* 488 and 594 fluorophores, developed by Molecular Probes, are much brighter than fluorescein, with higher quantum yield, lower background binding, improved solubility and more consistent fluorescence behavior across a wider pH range. Alexa Fluor®* 488 uses the same filter set as fluorescein. With Alexa Fluor®* 594, you can differentiate a FluoroNanogold-labeled target from a second target labeled with fluorescein, Alexa Fluor®* 488, green fluorescent protein, or other fluorophores.

  • Try blocking with 5% nonfat dried milk. This was found to be particularly effective when 1 to 5% nonfat dried milk was mixed with the incubation buffer and added to the specimen with the FluoroNanogold conjugate. Cold-water fish gelatin may also be helpful.

  • You may increase signal by using a tertiary labeling method: use your primary antibody, then detect with a biotinylated secondary antibody followed by FluoroNanogold-streptavidin. Tertiary labeling systems have been found to increase detection sensitivity in immunogold detection.

  • To obtain the cleanest gold labeling in the electron microscope, use a sodium citrate buffer wash after FluoroNanogold application, but before silver enhancement. 0.02 M sodium citrate at pH 7.0 works well with HQ Silver, while pH 3.5 works best with the Danscher silver formulation.

    Reduce hydrophobic interactions. Both the gold and fluorescent labels have some hydrophobicity: therefore, including in the wash buffer agents that reduce hydrophobic interactions may help to reduce non-specific binding. Suitable reagents include:

The following references provide helpful protocols for FluoroNanogold labeling:

  • Takizawa, T., and Robinson, J. M.: Correlative Microscopy of Ultrathin Cryosections is a Powerful Tool for Placental Research. Placenta, 24, 557-565 (2003).

  • Takizawa, T., and Robinson, J. M.: Ultrathin Cryosections. An important tool for immunofluorescence and correlative microscopy. J. Histochem. Cytochem., 51, 707-714 (2003).

More information:

*Alexa Fluor is a trademark of Molecular Probes, Inc.

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Labeling Other Functional Groups: Aldehydes and Ketones

If your molecule contains only aldehydes or ketones - no amines, thiols, or carboxyls - you are actually better off than those of you stuck with only hydroxyls, because carbonyl groups will react directly with primary amines - such as those on Monoamino Nanogold®.

Actually, the procedure for this is described on our web site, in the application note on labeling RNA and glycoproteins. In this procedure, the compound to be labeled is a glycoconjugate that has been oxidized using periodate to yield a dialdehyde: this is then reacted directly with Monoamino Nanogold. An important consideration is that the conjugation reaction yields a Schiff base, which must then be reduced to a secondary amine. An example is shown below.

[Nanogold-Aldehyde Labeling (3k)]

Example showing labeling of an aldehyde with Monoamino Nanogold

Example procedure:

  1. Dissolve 30 nmol of Monoamino Nanogold in 100 microliters anhydrous dimethyl sulfoxide (DMSO). Mix Nanogold-DMSO solution (16.7 microliters, 10-fold excess) in 100 mM PIPES (30 microliters) with oxidized biomolecule in buffer solution at pH 7.2 or higher (100 microliters). Incubate at 4°C for 60 minutes.

  2. Add 1.5 microliters of fresh 20 mg/mL borane tert-butylamine complex (stable for about 15 minutes). incubate at 4°C for 30minutes. Stop reaction with 2 microliters of acetone. After 3 minutes at 4°C, separate labeled biomolecule using an appropriate gel filtration column.

Alternatively, it may be possible to convert the aldehyde, or even the ketone, to a carboxylic acid. If so, you can then convert the carboxylic acid to a reactive ester using either 1-Ethyl-3-[3-dimethylaminopropyl] carbodiimide Hydrochloride (EDC) with Sulfo-N-hydroxysuccinimide or N,N-carbonyldiimidazole in DMF, then react the activated ester with Monoamino Nanogold.

You can also cross-link directly using one of the many carbonyl-reactive hydrazido- maleimide cross-linkers that 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.

More information:

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Nanogold® Cryo-EM Yields Structure of Enhancer-Binding Protein

Nanogold® can be used to label proteins and protein complexes with sufficient regularity and specificity that, when used with high-resolution electron microscopic and image analysis methods, the label can provide the orientation necessary to solve the structure. Rappas and collaborators provided an indirect application of this in their recent report describing the 20 angstrom resolution structure of a bacterial sigma54RNA polymerase holoenzyme activator, phage shock protein F [PspF(1-275)], which is bound to an ATP transition state analog in a complex with its basal factor, sigma54.

Bacterial RNA polymerase (RNAP) containing the sigma54 factor requires specialized transcriptional activator proteins, bacterial enhancer-binding proteins (EBPs), that interact with the basal transcription complex from remote DNA sites by DNA looping. EBPs bind upstream activating sequences via their C-terminal DNA binding domains, then form oligomers that use ATP hydrolysis to activate transcription. The central sigma54-RNAPinteracting domain of EBPs is responsible for ATPase activity and transcription activation; it belongs to the larger AAA+ (ATPase associated with various cellular activities) family of proteins. EBPs include phage shock protein F (PspF), nitrogen-fixation protein A (NifA), nitrogen-regulation protein C (NtrC), and C4-dicarboxylic acid transport protein D (DctD).

In order to elucidate the activation process, a structure-function analysis of one such system was conducted, comprising (i) a cryo-EM reconstruction of PspF_s AAA+ domain (residues 1 to 275, PspF(1-275)) in complex with sigma54 at the point of ATP hydrolysis (mimicked by in situ formed ADP.AlFx), (ii) the crystal structure of nucleotide-free (apo) PspF(1-275) at 1.75 Å resolution, and (iii) mutational analysis. PspF from Escherichia coli forms a stable oligomeric complex with sigma54 at the point of ATP hydrolysis. The highly conserved and EBP-specific GAFTGA amino acid motif is a crucial mechanical determinant for the successful transfer of energy from ATP hydrolysis in EBPs to the RNAP holoenzyme via the small N-terminal EBP-interacting domain of sigma54 (region I, ~56 residues). Nanoelectrospray mass spectroscopy of the PspF(1-275)sigma54 complex with ADP.AlFx established that six monomers of PspF(1-275) are in complex with a monomeric sigma54. Next, to confirm the presence and integrity of sigma54 in the particles, the N- and C- termini of sigma54 were labeled with Nanogold. Single cysteine substitutions were added to a cysteine-free sigma54 template in order to attach gold particles to specific domains: one cysteine was introduced in the N-terminal Region I domain (residue 46) while another construct had the single cysteine introduced in the C-terminal domain (residue 474). The single-cysteine E46Csigma54 and 474Csigma54 constructs were reacted overnight at 4°C with Monomaleimido Nanogold. Excess Nanogold was removed by gel filtration on a 24 mL Superdex 200 HR 10/30 column eluted with 200 mM sodium phosphate, pH 7.4, with 150 mM NaCl. The Nanogold-labeled constructs were then used to form the PspF(1-275)-ADP.AlFxsigma54 complex. Nanogold-labeled complexes were diluted to ~20 micrograms/mL in 200 mM sodium phosphate pH 7.4 with 150 mM NaCl, stained with 2% uranyl acetate, and air dried. Detection of Nanogold in both experiments confirmed the presence and the integrity of sigma54 in the complex.

Cryo-electron microscopy and image analysis of 9380 frozen hydrated PspF(1-275)-ADP.AlFx-sigma54 complexes was then used to solve the crystal structure of PspF(1-275). By fitting this crystal structure into the EM map, two loops involved in binding sigma54 were identified. Comparing enhancer-binding structures in different nucleotide states, combined with mutational analysis, support the proposition that nucleotide-dependent conformational changes occur that free the loops for association with sigma54.


Rappas, M.; Schumacher, J.; Beuron, F.; Niwa, H.; Bordes, P.; Wigneshweraraj, S.; Keetch, C. A.; Robinson, C. V.; Buck, M., and Zhang, X.: Structural insights into the activity of enhancer-binding proteins. Science, 307, 1972-1975 (2005).

More information:

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Nanogold® is for SEM Labeling, too!

We reported a while ago on the use of Nanogold® labeling for correlative fluorescence and scanning electron microscopy (SEM). This labeling method is further explored, and compared with alternatives, in a recent report by Wanner and co-workers. They find that currently, compared with other methods, SEM gives the highest resolution for the investigation of three-dimensional mitotic plant chromosome architecture, and specific chromatin staining techniques making use of simultaneous detection of back-scattered electrons and secondary electrons have provided conclusive information on the distribution of DNA and protein in barley chromosomes through mitosis.

Barley chromosomes were isolated and mounted either on laser marked glass slides using the "drop/cryo" technique, or on standard glass slides using the "suspension" technique. Chromosome control slides were subsequently fixed in 2.5% glutaraldehyde or 2% formaldehyde in cacodylate buffer (75 mM, pH 7). Slides for immunolabeling were incubated in phosphate-buffered saline (PBS: 0.13 M NaCl, 7 mM Na2HPO4, 3 mM NaH2PO4, pH 7.0, with 0.1% Tween 20). Cryo fracture of barley root tips was performed according to Tanaka (1980), modified by replacing dimethylsulfoxide with dimethylformamide and omitting osmium tetroxide thiocarbohydrazide impregnation cycles. For detection of DNA, root tips were stained with platinum blue, an oligomer of bis(acetonitrile)-platinum [(CH3CN)2Pt]n, for 1 hour after glutaraldehyde fixation. controlled decondensation experiments were conducted at room temperature using (i) citrate buffer (60 mM Na citrate, 15 mM CaCl2, pH 7.2) for 60 min; (ii) Tris/HCl buffer (10 mM, pH 7.2), (iii) 1% dextran sulfate in distilled water; and (iv) cacodylate buffer (75 mM, pH 7). For proteinase K treatment, chromosomes were fixed with glutaraldehyde (2.5% in 75 mM cacodylate buffer) then treated with proteinase K (1 mg/ml) for 2 hours at 37°C. For separate visualization of DNA, chromosomes were stained for 30 minutes at room temperature with platinum blue; DNase treatment was performed prior to glutaraldehyde fixation. Proteins were separately visualized after staining for 12 h at 60°C with 20% aqueous silver nitrate solution or with an aqueous solution of colloidal silver (0.5 g silver nitrate dissolved in 1.5 ml water gradually added to 25 ml of an aqueous solution of 0.25 % tannic acid and 2% sodium carbonate) containing 0.1 M of elementary silver at pH 8.

Labeling was done at room temperature; all wash steps were 3 x 10 min each. Slides were blocked with 1% bovine serum albumin in PBS for 30 min, then incubated with primary antibody, (polyclonal rabbit antibody against histone H3 phosphorylated at serine position 10) diluted 2 : 500 in the blocking solution for one hour. After washing in PBS, the secondary antibody (Nanogold-Fab' anti-rabbit IgG diluted at 1 : 20 in blocking buffer) was applied for one hour; slides were subsequently washed in PBS, and specimens were routinely postfixed with 2% glutaraldehyde in PBS (without Tween 20). Labeled specimens were washed with distilled water and enhanced using HQ Silver. Preparations exclusively examined in the secondary electron (SE) mode were sputter-coated with platinum, and those used for backscattered electron (BSE) detection (stained and immunolabeled specimens) were carbon-coated by evaporation, in both cases to a layer of 35 nm, and examined at an accelerating voltage of 8 kV (exclusively SE images) or 1230 kV (simultaneous SE and BSE imaging).

Applied to investigate the structural effects of different preparative procedures, these techniques supported the development of the "dynamic matrix model" for chromosome condensation. This postulates an energy-dependent process of looping and bunching of chromatin coupled with attachment to a dynamic matrix of associated protein fibers. Data from SEM analysis shows basic higher order chromatin structures: chromomeres and matrix fibers. Visualization of Nanogold-labeled phosphorylated histone H3 (ser10) with high resolution on chromomeres shows that functional modifications of chromatin can be located on structural elements in a 3D context.


Wanner, G.; Schroeder-Reiter, E., and Formanek, H.: 3D analysis of chromosome architecture: advantages and limitations with SEM. Cytogenet. Genome Res., 109 70-78 (2005).

Reference for cryo fracture technique:

Tanaka, K.: Scanning electron microscopy of intracellular structures. Int. Rev. Cytol., 68, 97125 (1980).

Reference for "drop/cryo" technique:

Martin, R.; Busch, W.; Herrmann, R. G., and Wanner, G.: Changes in chromosomal ultrastructure during the cell cycle. Chromosome Res., 4, 288-294 (1996).

Reference for "suspension" technique:

Schubert, I.; Dolezel, J.; Houben, A.; Scherthan, H., and Wanner, G: Refined examination of plant metaphase chromosome structure at different levels made feasible by new isolation methods. Chromosoma, 102, 96101 (1993).

More information:

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Other Recent Publications

Chen and co-workers describe the use of gold nanocages as optical imaging contrast agents in their recent article in Nano Letters. Gold nanocages less than 40 nm in diameter were prepared from silver nanocubes using the galvanic replacement reaction. Typically, a 75-microliter aliquot of a silver nanocube dispersion (8.1 mM in terms of silver) was added to a boiling solution of 5 mg poly(vinyl pyrridone) in 5 mL of deionized water: this diluted dispersion of silver nanocubes was then refluxed for 2 minutes. 750 microliters of 0.2 mM HAuCl4 aqueous solution was added using a microsyringe pump at a rate of 0.8 mL/min, and the solution refluxed for another 10 minutes with vigorous stirring until its color became stable. After cooling to room temperature, the white AgCl precipitate was dissolved by adding saturated NaCl, the mixture centrifuged at 10,000 rpm for 15 min, and the supernatant decanted. The product was then rinsed with water and centrifuged twice more, then re-dispersed in water for use. The edge length and wall thickness of these nanocages (calculated from TEM images) of 36.7 ± 3.5 and 3.3 ± 0.2 nm, respectively were close to values estimated from the stoichiometry for the replacement reaction. By controlling the molar ratio between silver and HAuCl4, the gold nanocages could be tuned to display surface plasmon resonance peaks in the range 400 to 1,000 nm. When tuned to around 800 nm, a wavelength commonly used in optical coherence tomography (OCT) imaging, OCT measurements on phantom samples yielded a moderate scattering cross-section of ~8.10 x 10-16 m2, but a very large absorption cross-section of ~7.26 x 10-15 m2 for these nanocages, suggesting their potential use as a new class of contrast agents for optical imaging. When conjugated to anti-HER2 antibodies (functionalized with succinimidyl propionyl poly(ethylene glycol) disulfide, or NHS-activated PEG, M.W. 1,109), the gold nanocages demonstrated for specific targeting of breast cancer cells.


Chen, J.; Saeki, F.; Wiley, B. J.; Cang, H.; Cobb, M. J.; Li, Z.-Y.; Au, L.; Zhang, H.; Kimmey, M. B.; Li, X., and Xia, Y.: Gold Nanocages: Bioconjugation and Their Potential Use as Optical Imaging Contrast Agents. Nano Lett., 5, 473-477 (2005).

Remember last month when we reported how Isherwood and Patel had used NanoVan with 10 nm colloidal gold labeling to map viral ultrastructure? This month, Holmgren and co-workers use our other negative staining reagent, Nano-W, in combination with FISH and immunohistochemistry, to find out the role of the minor capsid protein of papillomaviruses, L2, in the viral lifecycle. This group used the organotypic (raft) culture system to recapitulate the full viral life cycle of the high-risk human papillomavirus HPV31, either wild type or mutant for L2. After transfection, the L2 mutant HPV31 genome was able to establish itself as a nuclear plasmid in proliferating populations of poorly differentiated (basal-like) human keratinocytes, amplify its genome to high copy number and support late viral gene expression. Finally, it induced the formation of virus particles, identified by negative staining using Nano-W, in human keratinocytes that had been induced to undergo terminal differentiation. Analysis of the virus particles generated showed an approximate 10-fold reduction in the amount of viral DNA encapsidated into L2-deficient virions, with a greater than 100-fold reduction in the infectivity of L2-deficient virus. The latter deficiency, which cannot be accounted for solely by the 10-fold decrease in encapsidation, supports a conclusion that L2 contributes to at least two steps in the production of infectious virus.


Holmgren, S. C.; Patterson, N. A.; Ozbun, M. A, and Lambert P. F.: The minor capsid protein L2 contributes to two steps in the human papillomavirus type 31 life cycle. J. Virol., 79, 3938-3948 (2005).

Weiss and co-workers provided us with some evidence for the shelf life of our products this month, with their report on the use of our 5 nm colloidal gold-anti-rabbit conjugate to stain Methionine-Rich Repeat Proteins (MRRPs) in microsomal membranes. In vitro transcribed and translated MRRPs were isolated by centrifugation at 110,000 x g (1 h, 4°C) in Tris with 100 mM NaCl, pH 7.9; the microsomal pellet was resuspended in the same buffer and centrifugation repeated to wash the membranes. Membranes were then resuspended in 200 microliters of phosphate-buffered saline (PBS) at pH 7.4. The resuspended pellets were divided evenly into two tubes, washed in 200 microliters PBS, and blocked by resuspension in 100 microliters of PBSBSA buffer (20 mM phosphate, 150 mM NaCl pH 7.4 plus 0.5% BSA, 0.1% gelatin, and 0.05% Tween 20) for 30 minutes with gentle agitation at room temperature. MRRP antiserum was added (final dilution 1:100) and incubated overnight with gentle agitation at 4°C. After centrifugation at 100,000 x g, membranes were washed twice by resuspension in 100 microliters of PBSBSA buffer for 5 min followed by centrifugation and resuspended in 100 microliters of PBSBSA buffer. 5 nm Colloidal gold-labeled anti-rabbit IgG was added (final dilution 1:50) and incubated for 2 hours with gentle agitation at room temperature. Labeled membranes were centrifuged at 100,000 x g, washed twice in PBSBSA buffer, resuspended in 30 microliters of PBS, then applied to carbon coated copper EM grid and negatively stained with 4 % w/v uranyl acetate for electron microscopy. We are impressed because our colloidal gold conjugates were withdrawn from sale in 2000 while we developed something better. We do, however, continue to provide technical support for this situation, and in case anyone needs help with other colloidal gold conjugates.


Weiss, J. L.; Evans, N. A.; Ahmed, T.; Wrigley, J. D.; Khan, S.; Wright, C.; Keen, J. N.; Holzenburg, A, and Findlay, J. B.: Methionine-rich repeat proteins: a family of membrane-associated proteins which contain unusual repeat regions. Biochim. Biophys. Acta, 1668, 164-174 (2005).

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