'Recent advances in gold technology have led to probes with improved properties and performance for cell biologists: higher labeling density, better sensitivity, and greater penetration into tissues. Gold clusters, such as the 1.4-nm Nanogold, are gold compounds that can be covalently linked to Fab'' antibody fragments, making small and stable ... More
Ordered nanoparticle arrays formed on engineered chaperonin protein templates.
'Traditional methods for fabricating nanoscale arrays are usually based on lithographic techniques. Alternative new approaches rely on the use of nanoscale templates made of synthetic or biological materials. Some proteins, for example, have been used to form ordered two-dimensional arrays. Here, we fabricated nanoscale ordered arrays of metal and semiconductor ... More
Use of nanogold- and fluorescent-labeled antibody Fv fragments in immunocytochemistry.
AuthorsRibrioux S, Kleymann G, Haase W, Heitmann K, Ostermeier C, Michel H
JournalJ Histochem Cytochem
PubMed ID8648079
'Recombinant antibody fragments are emerging as a versatile tool in both basic research and medical therapy. We describe the procedures for direct labeling of engineered antibody fragments (Fv) with fluorescein or nanogold and their use in fluorescence and immunoelectron microscopy, respectively. The Fv fragments were produced in Escherichia coli, purified ... More
Localization of the N-terminal domain of the low density lipoprotein receptor.
AuthorsJeon H, Shipley GG
JournalJ Biol Chem
PubMed ID10889195
'The low density lipoprotein (LDL) receptor is a transmembrane glycoprotein performing "receptor-mediated endocytosis" of cholesterol-rich lipoproteins. At the N terminus, the LDL receptor has modular cysteine-rich repeats in both the ligand binding domain and the epidermal growth factor (EGF) precursor homology domain. Each repeat contains six disulfide-bonded cysteine residues, and ... More
Metallosomes.
AuthorsHainfeld JF, Furuya FR, Powell RD
JournalJ Struct Biol
PubMed ID10527904
'Structures and ordered arrays containing organometallic particles have potential application in nanofabrication, smaller computer components, optical devices, sensors, and membrane probes and as detection agents. Here, we describe construction of gold clusters covalently attached to lipids and their use in forming typical lipid structures: micelles, liposomes ("metallosomes"), and sheets on ... More
Vesicle-reconstituted low density lipoprotein receptor. Visualization by cryoelectron microscopy.
AuthorsJeon H, Shipley GG
JournalJ Biol Chem
PubMed ID10889196
'The low density lipoprotein (LDL) receptor is a key protein for maintaining cellular cholesterol homeostasis by binding cholesterol-rich lipoproteins through their apoB and apoE apoproteins. The LDL receptor is a transmembrane glycoprotein of M(r) approximately 115 kDa; based on its primary sequence, five distinct structural domains have been identified (Yamamoto, ... More
Organization of 'nanocrystal molecules' using DNA.
'Patterning matter on the nanometre scale is an important objective of current materials chemistry and physics. It is driven by both the need to further miniaturize electronic components and the fact that at the nanometre scale, materials properties are strongly size-dependent and thus can be tuned sensitively. In nanoscale crystals, ... More
Three-dimensional reconstruction of a complex of human alpha 2-macroglobulin with monomaleimido Nanogold (Au1.4nm) embedded in ice.
AuthorsBoisset N, Grassucci R, Penczek P, Delain E, Pochon F, Frank J, Lamy JN
JournalJ Struct Biol
PubMed ID1283687
'Cysteine 949 and glutamine 952 are known to be part of the thiol ester site of each of the four subunits of human alpha 2-macroglobulin (alpha 2M). The hydrolysis of this thiol ester bound to methylamine results in the incorporation of the amine and liberation of a free sulfhydryl group ... More
Chromatin condensation, cysteine-rich protamine, and establishment of disulphide interprotamine bonds during spermiogenesis of Eledone cirrhosa (Cephalopoda).
AuthorsGimenez-Bonafé P, Ribes E, Sautière P, Gonzalez A, Kasinsky H, Kouach M, Sautière PE, Ausió J, Chiva M
JournalEur J Cell Biol
PubMed ID12113475
'During spermiogenesis in Eledone cirrhosa a single protamine substitutes for histones in nuclei of developing spermatids. This protein displays a peculiar primary structure. It contains 22.6 mol% cysteine residues (19 cysteines in 84 residues). This makes it the most cysteine-rich protamine known. The proportion of basic residues is relatively low ... More
Labelling and characterisation of gamma-aminobutyric acidA receptor subunit-specific antibodies with monomaleimido Nanogold.
AuthorsRayner SL, Stephenson FA
JournalBiochem Soc Trans
PubMed ID9388760
Three-dimensional reconstruction of human alpha 2-macroglobulin and refinement of the localization of thiol ester bonds with monomaleimido nanogold.
AuthorsBoisset N, Penczek P, Pochon F, Frank J, Lamy J
JournalAnn N Y Acad Sci
PubMed ID7524399
Single-mismatch detection using gold-quenched fluorescent oligonucleotides.
AuthorsDubertret B, Calame M, Libchaber AJ
JournalNat Biotechnol
PubMed ID11283596
Here we describe a hybrid material composed of a single-stranded DNA (ssDNA) molecule, a 1.4 nm diameter gold nanoparticle, and a fluorophore that is highly quenched by the nanoparticle through a distance-dependent process. The fluorescence of this hybrid molecule increases by a factor of as much as several thousand as ... More
Labeling with nanogold and undecagold: techniques and results.
AuthorsHainfeld JF
JournalScanning Microsc Suppl
PubMed ID9601549
A significant new development in gold labeling for microscopy has been achieved through the use of gold cluster compounds that are covalently attached to antibodies or other probe molecules. These unique gold probes are smaller than most colloidal gold conjugates and exhibit improved penetration into tissues, higher labeling densities, and ... More
Conformational constraints in protein degradation by the 20S proteasome.
AuthorsWenzel T, Baumeister W
JournalNat Struct Biol
PubMed ID7773788
Conformationally stabilized peptides and unfolding intermediates of bovine alpha-lactalbumin have been used to define the degree of unfolding required for degradation by 20S proteasomes. It appears that complete unfolding and the absence of disulphide bonds are prerequisites for degradation, suggesting that a relatively narrow opening controls access to the inner ... More
Monomaleimidogold labeling of the gamma subunit of the Escherichia coli F1 ATPase examined by cryoelectron microscopy.
AuthorsWilkens S, Capaldi RA
JournalArch Biochem Biophys
PubMed ID1444442
A novel approach for locating sites of interest in a protein complex has been developed using monomaleimidonanogold (MMN). The Escherichia coli F1 ATPase, when prepared without the delta subunit, contains only a single reactive cysteine on one of the three copies of the alpha subunit. This site was reacted with ... More
Subcellular localization of estradiol receptor in MCF7 cells studied with nanogold-labelled antibody fragments.
AuthorsKessels MM, Qualmann B, Thole HH, Sierralta WD
JournalEur J Histochem
PubMed ID10068898
Ultrastructural localization studies of estradiol receptor in hormone-deprived and hormone-stimulated MCF7 cells were done using F(ab') fragments of three different antibodies (#402, 13H2, HT277) covalently linked to nanogold. These ultra-small, non-charged immunoreagents, combined with a size-enlargement by silver enhancement, localized estradiol receptor in both nuclear and cytoplasmic areas of non-stimulated ... More
Ni-NTA-gold clusters target His-tagged proteins.
AuthorsHainfeld JF, Liu W, Halsey CM, Freimuth P, Powell RD
JournalJ Struct Biol
PubMed ID10527908
Addition of six histidines to recombinant proteins has proved useful in their purification by nickel-affinity columns. This technology was adapted by synthesizing the chelator for nickel (nitrilotriacetic acid, NTA) onto the surface of gold clusters. These Ni-NTA-gold clusters were shown to specifically target the 6His region of tagged proteins. Results ... More
Immunogold labelling of neuroendocrine peptides with special reference to antibody specificity and multiple staining techniques.
AuthorsLarsson LI
JournalHistochem Cell Biol
PubMed ID8858369
Immunogold methods have been very important for research on the neuroendocrine system. The compatibility of immunogold probes with optimal contrasting for electron microscopy has made localizations of neuroendocrine peptides to different subtypes of secretory organelles possible and, currently, methods using covalent attachment of nanogold particles to antibodies and neuropeptide ligands ... More
Cryoelectron microscopy of low density lipoprotein in vitreous ice.
AuthorsSpin JM, Atkinson D
JournalBiophys J
PubMed ID7612855
In this report, images of low density lipoprotein (LDL) in vitreous ice at approximately 30 A resolution are presented. These images show that LDL is a quasi-spherical particle, approximately 220-240 A in diameter, with a region of low density (lipid) surrounded by a ring (in projection) of high density believed ... More
A covalent fluorescent-gold immunoprobe: simultaneous detection of a pre-mRNA splicing factor by light and electron microscopy.
Immunoprobes that combine a fluorescent label with a 1.4-nm gold cluster compound have been prepared by covalent conjugation with polyclonal antibody Fab' fragments. These new immunoconjugates allow the collection of two complementary sets of data, from fluorescence and electron microscopy, from a single labeling experiment. We find that incorporation of ... More
Structure of the Escherichia coli ATP synthase and role of the gamma and epsilon subunits in coupling catalytic site and proton channeling functions.
AuthorsCapaldi RA, Aggeler R, Gogol EP, Wilkens S
JournalJ Bioenerg Biomembr
PubMed ID1429536
The structure of the Escherichia coli ATP synthase has been studied by electron microscopy and a model developed in which the alpha and beta subunits of the F1 part are arranged hexagonally (in top view) alternating with one another and surrounding a central cavity of around 35 A at its ... More
Bidirectional amyloid fiber growth for a yeast prion determinant.
The polymerization of many amyloids is a two-stage process initiated by the formation of a seeding nucleus or protofibril. Soluble protein then assembles with these nuclei to form amyloid fibers. Whether fiber growth is bidirectional or unidirectional has been determined for two amyloids. In these cases, bidirectional growth was established ... More
Combined fluorescent and gold immunoprobes: reagents and methods for correlative light and electron microscopy.
AuthorsPowell RD, Halsey CM, Hainfeld JF
JournalMicrosc Res Tech
PubMed ID9712158
Immunoprobes which incorporate both a fluorescent label and a 1.4 nm gold cluster compound were prepared by covalent conjugation to Fab' antibody fragments of the Nanogold cluster label followed by a fluorescent moiety. These new immunoconjugates allow the collection of two complementary sets of data, from fluorescence and electron microscopy, ... More
Cytoplasmic intermediate filaments are stably associated with nuclear matrices and potentially modulate their DNA-binding function.
AuthorsTolstonog GV, Sabasch M, Traub P
JournalDNA Cell Biol
PubMed ID12015898
The tight association of cytoplasmic intermediate filaments (cIFs) with the nucleus and the isolation of crosslinkage products of vimentin with genomic DNA fragments, including nuclear matrix attachment regions (MARs) from proliferating fibroblasts, point to a participation of cIFs in nuclear activities. To test the possibility that cIFs are complementary nuclear ... More
Molecular immunolabeling with recombinant single-chain variable fragment (scFv) antibodies designed with metal-binding domains.
AuthorsMalecki M, Hsu A, Truong L, Sanchez S
JournalProc Natl Acad Sci U S A
PubMed ID11756693
To study the molecular structure and function of gene products in situ, we developed a molecular immunolabeling technology. Starting with cDNA from hybridomas producing monoclonal antibodies against biotin, catalase, and superoxide dismutase, we bioengineered recombinant single-chain variable fragment antibodies (scFv) and their derivatives containing metal-binding domains (scFv:MBD). As tested with ... More