A simple preparative technique for observation of microorganisms from municipal refuse by transmision electron microscopy

A simple preparative technique was developed to permit an observation of indigenous microorganisms from municipal refuse in their complex micro-environments by transmission electron microscopy. The principles of this method include a collection of microorgansims on TEM grids by centrifugation and their staining in chambers of the Beckman Airfuge centrifugal rotor. In electron micrographs, both intact microbial cells and non-biotic materials could be observed almost without optical interference.     

Determination of the molecular weight of proteins in heterogeneous mixtures: use of an air-driven ultracentrifuge for the analysis of protein--protein interactions.

A high-speed air-driven ultracentrifuge (Airfuge) has been used to study the molecular weights of proteins in heterogeneous mixtures. The method is based on previous studies (M. A. Bothwell, G. J. Howlett, and H. K. Schachman, 1978, J. Biol. Chem., 253, 2073–2077) which showed that at sedimentation equilibrium in the Airfuge the fraction of a protein remaining in an upper fraction of the Airfuge tube is almost linearly related to the exponential of the reduced molecular weight of the protein. In this study the total fraction of each particular protein remaining in an upper fraction of the Airfuge tube is determined by quantitative sodium dodecyl sulfate-gel electrophoresis. This procedure allows a wide range of proteins to be analyzed in a single Airfuge experiment. The method yields the “native” molecular weights of the protein components and is independent of the shape of the macromolecules being studied. Interactions occurring between the components in solution can be detected from the Airfuge data, and procedures are described which allow the experimental data for such interactions to be analyzed in terms of an equilibrium constant for the interaction. Results obtained for the electrostatic interaction at neutral pH between lysozyme and ovalbumin (K = 1.1 × 10⁵, m^?1) and lysozyme and bovine serum albumin (K = 1.0 × 10⁵, m^?1) agree well with literature values.     

An airfuge centrifugation procedure for the measurement of ligand binding to membrane-associated and detergent-solubilized plasma membrane receptors

A method is described in which high-speed centrifugation of membranes through an oil phase is used to separate membrane-bound and detergent-solubilized polypeptide receptor-iodinated ligand complexes from unbound ligands. Three centrifuges, the Brinkmann Eppendorf (5412), the Beckman Microfuge B and the Beckman Airfuge were evaluated for this capability. Under the conditions described, the Beckman Airfuge surpassed the others in recovering previously 125I- and 32P-labelled cell membranes. The Airfuge method was compared with the more classically employed membrane filtration method to measure specific [125I]insulin and [125I]thrombin binding to human placental membranes and an enriched plasma membrane fraction from mouse embryo fibroblasts, respectively, are found to be 4 to 6 times more sensitive. For example, specific binding of ligand to its receptor was demonstrated with 5 micrograms of protein. With slight modifications, the polyethyleneglycol 6000 method of precipitating 125I-labelled ligand-soluble receptor complexes can be adapted to the Airfuge sedimentation through oil procedure.     

A Rapid Method of Isolation and Fractionation of Proteoglycans Using a Vertical Tube Rotor for Isopycnic Centrifugation

A comparison was made between a vertical tube rotor and a fixed angle rotor for isopycnic centrifugation of proteoglycans. In the vertical tube rotor, isopycnic gradient was achieved much faster than in the conventional fixed angle rotor. The use of a vertical tube rotor for isopycnic centrifugation shortens the time considerably for the isolation of proteoglycans fron various tissues.     

Characterization of reacting solutions of macromolecules by laser light scattering and Airfuge centrifugation

Simple, fast and accurate measurements combining centrifugation in a table top Airfuge and laser light scattering in the Airfuge tube are described. The procedure achieves quantitative separation of particles according to their sedimentation coefficient in microliter volumes. By scanning through the sedimenting boundary association equilibrium constants are evaluated.     

Zonal Centrifuge Applied to the Purification of Herpesvirus in the Lucké Frog Kidney Tumor

A series of "winter" and "summer" Lucké kidney tumors of the frog (Rana pipiens) were homogenized and fractionated by differential centrifugation into nuclear, mitochondrial, and mitochondrial supernatant fractions. Winter tumors often contained high concentrations of herpesvirus, whereas no virus was observed in any of the summer tumors. The crude tumor fractions were further purified by rate-zonal sucrose gradient centrifugation in a B-XV zonal rotor. Gradient fractions rich in an enveloped, nucleated form of the herpesvirus from certain winter tumors have induced renal tumors when injected into developing frog embryos. Zonal centrifugation was followed by isopycnic banding of the virus zones for further purification of the different morphological forms of the virus.     

Rapid purification of photosystem I chlorophyll-binding proteins by differential centrifugation and vertical rotor

Photosystem I (PSI), which consists of a core complex and light-harvesting complex I (LHCI), is an important multisubunit pigment-protein complex located in the photosynthetic membranes of cyanobacteria, algae and plants. In the present study, we described a rapid method for isolation and purification of PSI and its subfractions. For purification of PSI, crude PSI was first prepared by differential centrifugation, which was applicable on a large scale at low cost. Then PSI was purified by sucrose gradient ultracentrifugation in a vertical rotor to reduce the centrifugation time from more than 20 h when using a swinging bucket rotor to only 3 h. Similarly, for subfractionation of PSI into the core complex and light-harvesting complex I, sucrose gradient ultracentrifugation in a vertical rotor was also used and it took only 4 h to obtain the PSI core, LHCI-680, and LHCI-730 at the same time. The resulting preparations were characterized by sodium dodecyl-sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), absorption spectroscopy, and 77 K fluorescence spectroscopy. In addition, their pigment composition was analyzed by high-performance liquid chromatography and the results showed that each Lhca could bind 1.5-1.6 luteins, 1.0 Violaxanthins, and 0.8-1.1 beta-carotenes on average, demonstrating that fewer carotenoids were released than with the slower traditional centrifugation. These results showed that the rapid isolation procedure, based on differential centrifugation and sucrose gradient ultracentrifugation in a vertical rotor, was efficient, and it should significantly facilitate preparation and studies of plant PSI. Moreover, the vertical rotor, rather than the swinging bucket rotor, may be a good choice for isolation of some other proteins.     

Isolation of Synaptic Complexes in a CsCl Gradient: Conditions for Maximal Resolution in the Zonal Rotor B-Xiv and Circular Dichroism Patterns

Synaptic complexes were isolated from different brain regions and developmental stages in a CsCl density gradient using a Ti-14 zonal rotor. The buoyant density of the synaptic complexes from all these tissues was 1.178–1.190. The conditions for maximal resolution were rapid displacement of the density gradient from the rotor (40 ml/min); continuous centrifugation of the particles in the gradient for 66 hours, sample loads not exceeding 200 mg membrane protein. The banding densities of the membranes in the CsCl gradient were shown to be a linear inverse function of their lipid content. The circular dichroism patterns of synaptic complexes and other neural membranes in suspension or SDS solutions were similar to those of membranes from other mammalian cells or from bacteria although the ellipticities of the neural membranes were lower. These studies indicate that the proteins in a variety of membranes are in an α-helical conformation.     

Experiments and theoretical calculations for forming gradients for zonal rotor centrifugation

A gradient mixer operating with a compressed inert gas instead of a pump is designed particularly for work with zonal rotors. Different exponential and linear profiles are calculated as they form along the rotor radius. The centrifugation time dependent on gradient shape and temperature is estimated.     

STUDIES ON ISOLATED CELL COMPONENTS : XVII. The Distribution of Cytochrome Oxidase Activity in Rat Liver Brei Fractionated in the Zonal Ultracentrifuge

The zonal ultracentrifuge was used to separate the subcellular components of rat liver brei into soluble phase, microsomal, mitochondrial, membranous fragments, and nuclear fractions during a single centrifugation. The centrifuge was run at 10,000 to 30,000 RPM for 15 to 240 minutes, and the rotor contained a 1200 ml sucrose gradient, varying linearly with radius from 17 to 55 per cent sucrose with a "cushion" of 66 per cent sucrose at the rotor edge. The distribution of the mitochondria was determined using cytochrome oxidase as the marker enzyme. An automated assay system for cytochrome oxidase was developed utilizing reduced cytochrome c as substrate, modules of the Technicon Autoanalyzer, and the Beckman DB Spectrophotometer. All of the cytochrome oxidase activity was restricted to a single peak in the gradient, and no activity could be detected in the zones occupied by the microsomes and nuclei. The mitochondrial fraction was isolated from rat liver brei in 0.25 M sucrose by differential centrifugation, and then run in the zonal ultracentrifuge.This fraction behaved in the zonal ultracentrifuge in the same way as mitochondria separated directly from intact brei. Observations of the isolated fractions in the phase contrast microscope indicated that a wide variety of granules was present in the mitochondrial zone in addition to the true mitochondria. Under the conditions employed, the mitochondria were sedimented essentially to their isopycnic position in the gradient at approximately 43.8 per cent sucrose, density 1.20 gm/cc.     

The isolation of coated vesicles from protoplasts of soybean

Fractions enriched in coated vesicles were obtained from protoplasts derived from suspension cultured Glycine max (L.) Merr. cells. Initial enrichment was achieved by isopycnic centrifugation of a protoplast homogenate through a linear sucrose gradient in a vertical rotor. The coated-vesicle fractions from this gradient were pooled and centrifuged through a second linear sucrose gradient in a rate zonal fashion to remove the larger contaminating membrane vesicles. The most prominent polypeptide in the coated-vesicle fractions, plant clathrin, had a relative molecular mass of approx. 190 kdalton as determined by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. Other enriched polypeptides included bands at 105, 100, 96, 64, 50, 38 and 32 kdalton. This method was compared with a procedure utilizing sucrose step gradients for preparing coated vesicles from soybean protoplasts. The effectiveness of the isopycnic-rate zonal centrifugation procedure was also tested for the preparation of bovine-brain coated vesicles.NRCC No. 23142     

Isolation of dna and rna from Streptococcus sobrinus OMZ176 using CsTFA gradients

• 1.1. A simple procedure for isolation of high molecular weight genomic DNA, and RNA, from Streptococcus sobrinus OMZ176 is described.
• 2.2. Cell cultures were grown aerobically for 10 hr.
• 3.3. Spheroplast formation and lysis was achieved by mutanolysin/lysozyme-dependent digestion of the cell wall, followed by N-lauroylsarcosinate-mediated lysis.
• 4.4. Nucleic acids were isolated directly from cell-lysates using cesium-trifluoroacetate (CsTFA) densitygradient centrifugation.
• 5.5. Three different centrifugation regimes were tested: self-generated density gradients in a fixed angle rotor; self-generated density-gradients in a swinging-bucket rotor; pre-formed density-gradients in a swinging-bucket rotor.
• 6.6. Genomic DNA isolated by the CsTFA-procedure was found to have higher purity as compared to genomic DNA isolated in a conventional CsCl gradient.
• 7.7. Isolated DNA was shown to be of a quality suitable for applications in molecular biology.

     

Purification of the protein crystal from Bacillus thuringiensis by zonal gradient centrifugation.

A method is described for the large-scale purification of the Bacillus thuringiensis protein crystal by zonal gradient centrifugation. NaBr gradients are employed in a Beckman J21-B centrifuge equipped with a JCF-Z rotor.     

Advantages and limitations of density gradient ultracentrifugation in the fractionation of human serum lipoproteins: role of salts and sucrose

Two density gradient ultracentrifugation methods, Redgrave et al. (1975. Anal. Biochem. 65: 42-49) and Nilsson et al. (1981. Anal. Biochem. 110: 342-348), currently used for the separation and analysis of plasma lipoproteins were compared with respect to their resolving power and capacity to obtain pure products as a function of time of ultracentrifugation using the same rotor (Beckman SW-40), speed (150,000 g), and temperature (14 degrees C). The effects of sucrose and salts were also investigated. The Redgrave gradient insured the separation of the major classes of plasma lipoproteins after 24 hr of centrifugation; however, equilibrium conditions were only reached after 48 hr, at which time the lipoproteins were contaminated by albumin. When the effluents from each rotor tube were continuously monitored at 280 nm, each lipoprotein band gave values that were higher than those from mass analyses. This was due to a light scattering effect, the extent of which was dependent on the concentration of lipoproteins and salts. Sucrose prevented the scattering effect and was found to bind irreversibly to the apolipoproteins. In contrast, after 66 hr centrifugation, the lipoproteins obtained from the Nilsson gradient exhibited a close correspondence between protein mass and absorbance values at 280 nm, had no scattering effect, and were uncontaminated by albumin. The difference in spectroscopic behavior between the Redgrave and the Nilsson procedures was attributed to three factors: 1) the presence of sucrose in the latter gradient and incorporation of this sugar into lipoproteins as assessed by mass and radioactivity measurements; 2), the salt density to which the serum samples were exposed to at the beginning of the ultracentrifugation; and 3) the final lipoprotein concentration.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification of very high density lipoproteins by differential density gradient ultracentrifugation

Differential density gradient ultracentrifugation procedures, utilizing a vertical rotor, were developed for the preparative purification of very high density lipoproteins (VHDL, density greater than 1.21 g/ml). The VHDLs of several insect species were purified as follows. An initial density gradient ultracentrifugation step removed lipoproteins of lower density from the VHDL-fraction, which partially separated from the nonlipoproteins present in the infranatant. A complete separation was achieved by a second centrifugation step employing a modified gradient system. The use of a vertical rotor and specially designed discontinuous gradients allows a relatively fast, efficient, and economical isolation of the class of very high density lipoproteins. Similar gradient systems should be useful for the detection and purification of VHDLs from other sources.     

An efficient preparative isopycnic flotation method for the isolation of chitosomes

Summary Chitin synthetase, a key enzyme in fungal cell wall biosynthesis, is located in chitosomes (microvesicles). To produce large quantities of chitosomes for immunochemical and biochemical characterization, we developed a two-step purification procedure in which isopycnic sucrose density gradients were centrifuged at ultra-high gravitational forces (fixed-angle rotor at 361,000×g R_av). Chitosomes from yeast cells ofMucor rouxii were separated from the soluble proteins and from the larger membranes by isopycnic centrifugation of the cell-free extract. The resulting crude chitosome sample was adjusted to a higher sucrose concentration, and a sucrose gradient was layered over the sample. Upon recentrifugation, the chitosomes moved up into the gradient and equilibrated at their buoyant density (1.15–1.16). This accelerated flotation separated contaminating particles of higher buoyant density (larger vesicles, ribosomes, and other miniorganelles) and yielded a large population of microvesicles with a mean diameter of 48.9±13.8 nm. This preparation contained vesicles essentially free of other particulate contaminants; more than 99% of the vesicles were smaller than 100 nm. When required, an additional velocity centrifugation step was added to remove the larger vesicles from the chitosome samples. This streamlined method for chitosome isolation was much simpler and faster than earlier isolation procedures, gave a high yield of functional chitosomes, and made the large scale isolation of these organelles possible.     

Evaluation of a robotic system for the recovery of peripheral blood mononuclear cells

Investigations into immune responses are often based upon recovery of peripheral blood mononuclear cells (PBMC). To this purpose, the recovery of PBMC by gradient centrifugation is labour-intensive and requires a reasonable level of skill by the laboratory technician. Thus, we set out to determine whether laboratory automation equipment could be used for the recovery of PBMC from blood samples of horses, pigs and cattle, based on the Ficoll-Paque gradient centrifugation technique. Mixing of blood samples with PBS, layering of diluted blood onto Ficoll-Paque gradients, recovery of separated PBMC in RPMI 1640 medium were performed using an automated robotic system, the SBF200 (AM Robotic Systems, Warrington, UK) under laminar air flow conditions. Tubes were tagged with bar codes and manually placed after gradient centrifugation into a tube reader to measure the volume and position of the PBMC layer. The results of the automated procedure compared very well to those of the manual one in terms of percent cell recovery, sterility and cell viability. Also, a high throughput of samples could be implemented: with the integration of cell counting it should be possible for 96 blood samples to be processed, including the production of aliquots, by one person in a day.     

A method for visualizing and quantifying the total complement of free and membrane-bound ribosomes in rat liver.

A method is described for both visualization and quantification of the total complement of rat liver free and membrane-bound ribosomes, undegraded by nucleolysis and unaggregated by pelleting. The method involves: (a) differential centrifugation of liver homogenate which separates free and membrane-bound ribosomes; (b) treatment of the fractions with detergents to solubilize membranes and remove nuclei; (c) centrifugation of a portion of each fraction to remove all the ribosomes; (d) sedimentation of the samples and blanks on sucrose gradients; and (e) difference photometric scanning of the gradients, sample minus ribosome-free blank, to detect the ribosomes free of interference from nonribosomal materials. The use of the SW 56 rotor in the initial centrifugation and of a high Mg²⁺ concentration (20 mm) in the medium used to suspend the bound fraction prior to detergent treatment were found to be essential in obtaining bound polysomes of large size (～19-somes). The difference scanning technique is shown to be a sensitive, accurate, and reproducible means of eliminating interference from nonribosomal materials, principally detergents and protein, and of quantifying ribosomes in both fractions. The method is rapid (3.5 h), simple to perform, and well suited for the analysis of multiple liver samples. It can be used to assess the concentration, distribution, organization, and average size of the total complement of rat liver free and membrane-bound ribosomes in a single experiment.     

Rapid Isolation of Metabolically Active Mitochondria from Rat Brain and Subregions Using Percoll Density Gradient Centrifugation

Two procedures are described for isolating free (nonsynaptosomal) mitochondria from rat brain. Both procedures employ a discontinuous Percoll gradient and yield well coupled mitochondria which exhibit high rates of respiratory activity and contain little residual contamination by synaptosomes or myelin. The procedures are considerably more rapid than methods described previously for the isolation of brain mitochondria and do not require an ultracentrifuge or swing-out rotor. The first method separates mitochondria by gradient centrifugation from a P2 (crude mitochondrial) fraction and is likely to be widely applicable for studies in which at least 500 mg of tissue are available as starting material. In the second method, the unfractionated homogenate is subjected directly to gradient centrifugation. This method requires the preparation of more gradients (per gram of tissue) than the first method and yields a subcellular fraction with slightly more synaptosomal contamination. However, this second procedure is more rapid, requires less manipulation of the tissue, and is suitable for obtaining mitochondria with well preserved metabolic characteristics from subregions of single rat brains.     

An automated method for determining buoyant density of nucleic acids using a preparative ultracentrifuge

We present a technique for analytical buoyant density sedimentation of nucleic acids which is performed in a preparative ultracentrifuge, in contrast to an analytical ultracentrifuge. Following centrifugation in a preparative rotor, small cylindrical quartz tubes are optically scanned; upon completion of the scan the data are processed immediately by a microcomputer and the buoyant density of the nucleic acid is calculated. Experimental data are presented employing several different deoxyribonucleic acids banded in neutral and alkaline cesium sulfate. Results are independent of rotor speed, location of bands within the gradient, and loading density of the cesium sulfate solution. Derived buoyant density values agree within 0.5% of previously published values.