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PubMed2003

Optimization of in-gel protein digestion system in combination with thin-gel separation and negative staining in 96-well plate format.

Improvement of in-gel digestion efficiency is highly desirable for one- or two-dimensional gel electrophoretic separation and mass spectrometric (MS) analysis in proteomics, because the resultant increases in sequence coverage and MS signal intensity lead to higher confidence in protein identification. Here an optimized in-gel digestion system, in combination with thin-gel separation and negative staining in a high-throughput format using 96-well plates, is described. The combination of negative staining and protein separation on a 0.9 mm thick gel showed a clear improvement in in-gel digestion efficiency in comparison with the more typical protocols such as the combination of silver staining and a 1.0 mm gel. In addition, the use of 96-well plates to increase throughput did not decrease the efficiency of this strategy when the stirring of the gel pieces in processes such as destaining, washing, gel-shrinking and peptide extraction was performed by sonication instead of shaking the plates. This procedure was optimized and applied to identify proteins of the postsynaptic density fraction; 105 proteins were identified after SDS-PAGE separation.

Animals↗
PubMed1992

Three-dimensional structure of the truncated core of the Saccharomyces cerevisiae pyruvate dehydrogenase complex determined from negative stain and cryoelectron microscopy images.

Dihydrolipoamide acyltransferase (E2), a catalytic and structural component of the three functional classes of multienzyme complexes that catalyze the oxidative decarboxylation of alpha-keto acids, forms the central core to which the other components are attached. We have imaged by negative stain and cryoelectron microscopy the truncated dihydrolipoamide acetyltransferase core (60 subunits; M(r) = 2.7 x 10(6)) of the Saccharomyces cerevisiae pyruvate dehydrogenase complex. Using icosahedral particle reconstruction techniques, we determined its structure to 25 A resolution. Although the model derived from the negative stain reconstruction was approximately 20% smaller than the model derived from the frozen-hydrated data, when corrected for the effects of the electron microscope contrast transfer functions, the reconstructions showed excellent correspondence. The pentagonal dodecahedron-shaped macromolecule has a maximum diameter, as measured along the 3-fold axis, of approximately 226 A (frozen-hydrated value), and 12 large openings (approximately 63 A in diameter) on the 5-fold axes that lead into a large solvent-accessible cavity (approximately 76-140 A diameter). The 20 vertices consist of cone-shaped trimers, each with a flattened base on the outside of the structure and an apex directed toward the center. The trimers are interconnected by 20 A thick "bridges" on the 2-fold axes. These studies also show that the highest resolution features apparent in the frozen-hydrated reconstruction are revealed in a filtered reconstruction of the stained molecule.

Acetyltransferases↗
PubMed1988

Ultrastructural detection of herpes-type virions by negative staining in oral hairy leukoplakia.

Hairy leukoplakia (HL) is a newly recognized virus-associated lesion of oral mucosa that occurs in persons infected with human immunodeficiency virus. Studies have demonstrated Epstein-Barr virus within epithelial cells of HL. The authors examined 12 cases of HL by transmission electron microscopy to compare the use of a negative staining technique versus routine plastic embedment for the detection of viruses. Herpes-type virions were identified by both methods in 11 cases. One case had negative results for viruses by both methods. Negative staining is a simple and rapid technique that compares favorably with plastic embedment in the detection of herpes-type virions in HL.

Acquired Immunodeficiency Syndrome↗
PubMed1981

Negatively stained punctated cell contacts between luteal cells in rabbit and rat.

Cell contacts from rat and rabbit luteal cells have been spread on phosphotungstic acid solution after tissue fractionation or spreading was accomplished by a direct technique avoiding homogenization. Sectioned material was also examined. It was shown by negative staining that the cell contacts were composed of a multitude of bridges. These bridges were either filamentous (condensed form) or clearly showed a larger (expanded form) central particle. The corresponding image in top view showed respectively small and larger granules without any particular arrangement. Large granular structures were also seen on single plasma membranes. Evidence is presented that the condensed and the expanded form are two aspects of the same junctional structure. It is also suggested that both aspects are related to the tightness of the contact. Up to now this type of cell contract has been called "septate-like". However because of the dotted appearance of these contacts after colloidal lanthanum and after negative staining the denomination "punctated cell contact" may be better adapted.

Animals↗
PubMed2004

Use of negative stain and single-particle image processing to explore dynamic properties of flexible macromolecules.

Flexible macromolecules pose special difficulties for structure determination by crystallography or NMR. Progress can be made by electron microscopy, but electron cryo-microscopy of unstained, hydrated specimens is limited to larger macromolecules because of the inherently low signal-to-noise ratio. For three-dimensional structure determination, the single particles must be invariant in structure. Here, we describe how we have used negative staining and single-particle image processing techniques to explore the structure and flexibility of single molecules of two motor proteins: myosin and dynein. Critical for the success of negative staining is a hydrophilic, thin carbon film, because it produces a low noise background around each molecule, and stabilises the molecule against damage by the stain. The strategy adopted for single-particle image processing exploits the flexibility available within the SPIDER software suite. We illustrate the benefits of successive rounds of image alignment and classification, and the use of whole molecule averages and movies to analyse and display both structure and flexibility within the dynein motor.

Animals↗
PubMed1983

Equivalence of the projected structure of thin catalase crystals preserved for electron microscopy by negative stain, glucose or embedding in the presence of tannic acid.

Thin crystals of beef liver catalase have been examined by electron microscopy following various preservation procedures. In the first part of this investigation, micrographs of three principal projections were obtained from thin sections of micro-crystals embedded in the presence of tannic acid. Computer reconstructions confirmed the space group assignment of P2(1)2(1)2(1) and permitted the packing arrangement of the catalase tetramers to be deduced to a resolution of about 20 A. These results corroborate the packing model for this crystal form proposed by Unwin (1975) on the basis of molecular modeling of one projection. In the second part of this investigation, the projected structures of the thin crystals in various preserving media were compared. The negative contrasting of crystals embedded in the presence of tannic acid was confirmed by direct comparison with non-embedded, negatively stained thin platelet crystals. In addition, good agreement at 20 A resolution was observed between the structure of negatively stained crystals and the structure of crystal platelets preserved in glucose and examined by low-dose methods, while moderate agreement was established with the published data of Taylor (1978) for crystals embedded in thin ice films. Tannic acid alone was also found to serve as a suitable medium for preserving catalase crystals to a resolution of 3 X 7 A as judged by electron diffraction. Overall, we demonstrate that projections obtained from thin sections of catalase crystals embedded in the presence of tannic acid can provide a reliable, negatively contrasted representation of the protein structure to 20 A resolution. Examination of sectioned crystals could thus provide a useful adjunct to X-ray crystallographic studies of protein crystals and three-dimensional reconstruction of crystal thin sections should ultimately be possible.

Animals↗
PubMed1969

Periplasmic structure of frozen-etched and negatively stained cells of Bacillus licheniformis as correlated with penicillinase formation.

Bacillus licheniformis strain 749/C (constitutive for penicillinase formation) and uninduced cells of strain 749 (penicillinase-inducible) were examined after freezeetching. In the early stationary phase, strain 749/C organisms had clusters of vesicles (30 to 40 nm in diameter) on the outer surface of the plasma membrane. These are randomly distributed on the membrane, including the region of septum formation. The vesicles are not intimately associated with the plasma membrane, and their inner and outer surfaces are devoid of particles. Periplasmic vesicles were not detected by freeze-etching in strain 749 (uninduced) or in young cells of 749/C; however, the membrane of mid-logarithmic phase 749/C cells had a corrugated appearance. Negatively stained 749/C cells (logarithmic phase) also showed many vesicular and tubular bodies in the periplasm as well as septal and cytoplasmic mesosomes of typical morphology. The periplasmic structures appear to be formed either by evagination of plasma membrane or by migration of vesicular bodies from the membranous pockets of the cytoplasm. Stationary phase cells of 749/C still have many periplasmic vesicular bodies; however, the mesosomes are greatly reduced both in number and size. In sharp contrast, strain 749 organisms have very few structures similar to the periplasmic bodies of strain 749/C. These findings support our previous view that penicillinase-producing cells of 749/C have periplasmic membranous structures that are rare in the uninduced strain 749, though there is some lack of correspondence between freeze-etching, negative staining, and thin section data. These structures may be important for the retention or storage of penicillinase in the cell.

Bacillus↗
PubMed1985

Negative staining of myosin molecules.

A reproducible method has been developed for the negative staining of myosin molecules. The dimensions of stained molecules are in close agreement with those obtained by metal shadowing. Sharp bends in the tail, indicative of hinge regions, were observed at two positions 44 nm and 76 nm from the head-tail junction. The tail was often ill-defined at the position of the first (44 nm) bend. The bend positions may be sites of proteolytic cleavage that result in the production of long and short myosin subfragment S2. About half the molecules exhibited bending to various degrees at one or both of these positions, but cases where the tail folded back on itself in a 180 degrees bend were comparatively rare (approximately equal to 10%). However, in the absence of EGTA, a large fraction of the molecules (approximately equal to 80%) exhibited 180 degrees bends. A small region, approximately 20 nm long, at the tip of the tail often appears to be significantly different from the rest. The heads are about 19 nm long and roughly pear-shaped. Although sometimes straight, more often they show a pronounced curvature. Both senses of curvature were observed, but those curved in a clockwise manner were the most common, indicating preferential binding of one side of the head to the carbon substrate. An analysis of the different combinations of head shapes in individual molecules indicates that each head can rotate independently around its long axis. No preferred angle of orientation between the two heads in a molecule, or between either head and the tail could be found. Substructure has been observed within the heads.

Animals↗
PubMed1980

The surface layer of Mycoplasma synoviae as demonstrated by the negative staining technique.

Four strains of Mycoplasma synoviae grown in broth medium were negatively stained and examined in the electron microscope. The morphology of all four strains was essentially similar to that of other mycoplasmas. Fixation of the mycoplasmas in hypertonic solutions resulted in extensive distortion of the organisms as did incubation for periods up to 50 h at 37 degrees C. No relationship could be demonstrated between the surface projections observed on the organisms and their ability to haemagglutinate chicken red blood cells.

Culture Media↗
PubMed1991

Vertebrate muscle Z-line structure: an electron microscopic study of negatively-stained myofibrils.

Structural features of the Z-lines of rabbit psoas muscle myofibrils have been studied in the electron microscope with a negative staining technique. The results obtained suggest the presence of about 20 nm periodicity in the structural organization of the Z-line region: a band pattern of five bands of extra density spaced about 20 nm apart was revealed in the Z-region and the Z-filaments connecting actin filaments from neighbouring sarcomeres often appeared to be positioned at intervals of 17-20 nm. An electron microscopic investigation of the interaction in vitro of two major Z-line proteins, alpha-actinin and F-actin, indicated that the positions of alpha-actinin bridges between actin filaments are defined by relative azimuthal positions of actin subunits. A possible arrangement of actin-linking macromolecular bridges in the Z-region is considered. It is supposed that the arrangement of the Z-filaments is related to the helical symmetry of actin-containing filaments. Also, the banded appearance of the Z-region is interpreted as arising from the arrangement of crossbridges connecting thin filaments of the same sarcomeres.

Actinin↗
PubMed1963

Some observations on negatively stained mitochondria.

A particulate component of mitochondria is described which is observed in negatively stained preparations of unfixed whole mitochondria or their fragments. It consists of a roughly spherical particle, approximately 85 A in diameter, found on the surface of the cristae and probably also on the side of the mitochondrial envelope which faces the matrix. It is attached to the underlying membrane by a narrow stalk 40 to 50 A long. The particle appears to be rather labile and it is destroyed or at least rendered unrecognizable by all conventional fixatives used in electron microscopy, especially OsO(4).

Coloring Agents↗
PubMed1994

Negative staining can cause clumping of Bordetella pertussis fimbriae.

The state of fimbriae type 2 (Fim 2) and fimbriae type 3 (Fim 3) preparations from Bordetella pertussis were examined by negative stain electron microscopy. Uranyl acetate induced clumping of Fim 3 regardless of pH and was unsuitable as a stain for establishing the state of fimbriae. Both ammonium molybdate and sodium phosphotungstate were able to show the differences in Fim 3 stored at pH 7.2 and pH 9.5.

Antibodies, Bacterial↗
PubMed1985

Nonfluorescent negative stain for alanopine dehydrogenase activity on starch gels.

A two-step procedure for negative staining NADH-dependent alanopine dehydrogenase (ALPDH) activity on starch gels is described, using nitroblue tetrasolium and phenasine methosulfate. This method gives a blue background with unreacted NADH, and white areas where ALPDH is located. Using this technique the first reliable evidence of the existence of true ALPDH isoenzymes is obtained.

Animals↗
PubMed2002

Three-dimensional architecture of the eukaryotic multisynthetase complex determined from negatively stained and cryoelectron micrographs.

This study provides the first description of the three-dimensional architecture of the multienzyme complex of aminoacyl-tRNA synthetases. Reconstructions were calculated from electron microscopic images of negatively stained and frozen hydrated samples using three independent angular assignment methods. In all cases, volumes show an asymmetric triangular arrangement of protein domains around a deep central cavity. The structures have openings or indentations on most sides. Maximum dimensions are ca. 19x16x10 nm. The central cavity is 4 nm in diameter and extends two-thirds of the length of the particle.

Amino Acyl-tRNA Synthetases↗
PubMed1993

Collagen-glutaraldehyde interaction as revealed by the D-banding of negatively stained fibrils and computer-drawn band patterns.

Band patterns exhibited under electron microscope by native collagen fibrils fixed with glutaraldehyde (2.5%-5% GA diluted in 0.1M phosphate buffer, pH 7.4) and negatively stained with phosphotungstic acid (1% PTA diluted in the same buffer) were digitized to form both bandings and microdensitometric traces. Collaterally, computer-drawn band patterns and traces were yielded on the basis of the "quarter stagger" model and primary structure of alpha 1(I) and alpha 2(I) tropocollagen chains and by selecting options related to specific collagen-GA interactions. Comparisons between actual and simulated patterns suggest that lysines and hydroxylysines should react with GA residues in a 1:3 ratio, while GA-reactivity of histidyl and tyrosyl residues seem to be excluded. On the other hand, an improvement of simulations was achieved by also selecting hydroxyprolines (in addition to lysines and hydroxylysines), which seemed to react with GA in a 1:1 ratio. Considering the bifunctionality of GA, it is suggested that during fixation, heteropolymers form, composed of GA-hexamers bonded to couples of lysyl and/or hydroxylysyl residues. The hypothesis is advanced of an additional formation of GA-dimers, each bonded to two hydroxyprolines.

Animals↗