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Sample records for agarose

  1. The relationship of agarose gel structure to the sieving of spheres during agarose gel electrophoresis.

    OpenAIRE

    Griess, G A; Guiseley, K B; Serwer, P

    1993-01-01

    To understand the organization of fibers in an agarose gel, digitized electron micrographs are used here to determine the frequency distribution of interfiber distance (2Pc) in thin sections of agarose gels. For a preparation of underivatized agarose, a 1.5% gel has a Pc distribution that is indistinguishable from the Pc distribution of a computer-generated, random-fiber gel; the log of the occurrence frequency (F) decreases linearly as a function of Pc. As the agarose concentration decreases...

  2. Agarose Gel Electrophoresis for the Separation of DNA Fragments

    OpenAIRE

    Lee, Pei Yun; Costumbrado, John; Hsu, Chih-Yuan; Kim, Yong Hoon

    2012-01-01

    Agarose gel electrophoresis is the most effective way of separating DNA fragments of varying sizes ranging from 100 bp to 25 kb1. Agarose is isolated from the seaweed genera Gelidium and Gracilaria, and consists of repeated agarobiose (L- and D-galactose) subunits2. During gelation, agarose polymers associate non-covalently and form a network of bundles whose pore sizes determine a gel's molecular sieving properties. The use of agarose gel electrophoresis revolutionized the separation of DNA....

  3. Electron beam sterilization of the agarose gel used for electrophoresis

    International Nuclear Information System (INIS)

    The results obtained by electron beam (EB) sterilization of the plates with agarose gel used for human serum protein electrophoresis are presented. Also, the results obtained by human serum protein electrophoresis performed with agarose gel plates irradiated at different EB doses, from 4 kGy to 20 kGy, are presented. The microbiological results demonstrate that above 5 kGy the irradiated agarose plates are sterile. The EB irradiation of the agarose gel plates in the dose range of 7-9 kGy gives the best results for both, sterilization and protein fraction separation processes. (author)

  4. Composition of agarose substrate affects behavioral output of Drosophila larvae

    Directory of Open Access Journals (Sweden)

    Anthi Aristomenis Apostolopoulou

    2014-01-01

    Full Text Available In the last decade the Drosophila larva has evolved into a simple model organism offering the opportunity to integrate molecular genetics with systems neuroscience. This led to a detailed understanding of the functional neuronal networks for a number of sensory functions and behaviors including olfaction, vision, gustation and learning and memory. Typically, behavioral assays in use exploit simple Petri dish setups with either agarose or agar as a substrate. However, neither the quality nor the concentration of the substrate is generally standardized across these experiments and there is no data available on how larval behavior is affected by such different substrates. Here, we have investigated the effects of different agarose concentrations on several larval behaviors. We demonstrate that agarose concentration is an important parameter, which affects all behaviors tested: preference, feeding, learning and locomotion. Larvae can discriminate between different agarose concentrations, they feed differently on them, they can learn to associate an agarose concentration with an odor stimulus and crawl faster on a substrate of higher agarose concentration. Additionally, we have investigated the effect of agarose concentration on three quinine based behaviors: preference, feeding and learning. We show that in all cases examined the behavioral output changes in an agarose concentration-dependent manner. Our results suggest that comparisons between experiments performed on substrates differing in agarose concentration should be done with caution. It should be taken into consideration that the agarose concentration can affect the behavioral output and thereby the experimental outcomes per se potentially due to an increased escape response on more rigid substrates.

  5. Uptake and Recovery of Lead by Agarose Gel Polymers

    Directory of Open Access Journals (Sweden)

    Anurag Pandey

    2009-01-01

    Full Text Available Problem statement: The uptake and recovery of lead ions were investigated by using agarose gel polymers. Approach: The experimental results showed that the agarose gel were effective in removing Pb (II from solution. Biosorption equilibrium was approached within 4 h. Pseudo second-order was applicable to all the sorption data over the entire time range. Results: The sorption data conformed well to both the Langmuir and the Freundlich isotherm model. The maximum adsorption capacity (qmax onto agarose gel was 115 mg g-1 for Pb (II. The maximum uptake of metal ions was obtained at pH 2.0. At temperature 35°C, the biosorption of metal ions was found to be highest, with increase or decrease in temperature resulted in a decrease in the metal ions uptake capacity. Conclusion: Elution experiments were carried out to remove Pb (II ions from loaded agarose gel and the bound metal ions could be eluted successfully using 0.1 M EDTA solution. The results suggest that agarose gel can be used as a biosorbent for an efficient removal of Pb(II ions from aqueous solution.

  6. Recovery of uranium by tannin immobilized on agarose

    International Nuclear Information System (INIS)

    Tannin, which is a ubiquitous and inexpensive material, was immobilized on agarose gel to produce an excellent adsorbent for uranium recovery from seawater. Optimal conditions for the immobilization of tannin on agarose gel by both the epichlorohydrin and cyanuric chloride coupling procedures were examined in detail. The resulting immobilized tannin has a highly selective ability to adsorb uranium and applicability in both column and batch systems. This adsorbent can recover uranium from natural seawater with high efficiency. The maximum adsorption capacities were 1850 μg uranium g-1 adsorbent for the tannin immobilized on agarose gel by the epichlorohydrin coupling procedure and 1062 μg g-1 adsorbent for that produced by the cyanuric chloride coupling procedure. (author)

  7. Optical investigation of diffusion of levofloxacin mesylate in agarose hydrogel

    Science.gov (United States)

    Tan, Shuaixia; Dai, Hongjun; Wu, Juejie; Zhao, Ning; Zhang, Xiaoli; Xu, Jian

    2009-09-01

    Real-time electronic speckle pattern interferometry method has been applied to study the diffusion behavior of levofloxacin mesylate (MSALVFX) in agarose hydrogel. The results show that the diffusivity of solute decreases with the increase of concentration of agarose and adapts to Kohlrausch's law. Furthermore, Amsden's model, based on the retardance effect associated with polymer chain flexibility, was employed to simulate the diffusion behavior. The consistent results suggest that the retardance effect dominates the diffusion process of MSALFVX in hydrogel; moreover, polymer chain flexibility greatly affects drug transport within the polymer matrix.

  8. Purification of the Rous sarcoma virus src kinase by casein-agarose and tyrosine-agarose affinity chromatography.

    OpenAIRE

    Fukami, Y.; Lipmann, F

    1985-01-01

    A simple and effective purification method for the src kinase, the transforming gene product of Rous sarcoma virus, has been developed by using affinity chromatography on casein-agarose and tyrosine-agarose columns. NaDodSO4/polyacrylamide gel electrophoresis and silver staining analysis showed that the purified kinase preparation was composed of a predominant polypeptide of 60,000-Da. In most of the preparations, however, three minor proteins (54,000, 52,000, and 15,000 Da) were also detecte...

  9. NEUTROPHIL MIGRATION UNDER AGAROSE IS ALTERED BY COPPER DEFICIENCY

    Science.gov (United States)

    Dietary copper deficiency alters neutrophil sequestration and margination within the pulmonary microcirculation in rats. In the current study, the role of copper in CD1 lb/CD18-dependent an -independent migration of neutrophils under agarose was studied. Male, weanling Sprague-Dawley rats were fed p...

  10. Electrophoresis of DNA in agarose gels, polyacrylamide gels and in free solution

    OpenAIRE

    Stellwagen, Nancy C.

    2009-01-01

    This review describes the electrophoresis of curved and normal DNA molecules in agarose gels, polyacrylamide gels and in free solution. These studies were undertaken to clarify why curved DNA molecules migrate anomalously slowly in polyacrylamide gels but not in agarose gels. Two milestone papers are cited, in which Ferguson plots were used to estimate the effective pore size of agarose and polyacrylamide gels. Subsequent studies on the effect of the electric field on agarose and polyacrylami...

  11. Electrophoresis and orientation of F-actin in agarose gels.

    OpenAIRE

    Borejdo, J; Ortega, H.

    1989-01-01

    F-Actin was electrophoresed on agarose gels. In the presence of 2 mM MgCl2 and above pH 8.5 F-actin entered 1% agarose; when the electric field was 2.1 V/cm and the pH was 8.8, F-actin migrated through a gel as a single band at a rate of 2.5 mm/h. Labeling of actin with fluorophores did not affect its rate of migration, but an increase in ionic strength slowed it down. After the electrophoresis actin was able to bind phalloidin and heavy meromyosin (HMM) and it activated Mg2+-dependent ATPase...

  12. Agarose and methylcellulose hydrogel blends for nerve regeneration applications

    Science.gov (United States)

    Martin, Benton C.; Minner, Eric J.; Wiseman, Sherri L.; Klank, Rebecca L.; Gilbert, Ryan J.

    2008-06-01

    Trauma sustained to the central nervous system is a debilitating problem for thousands of people worldwide. Neuronal regeneration within the central nervous system is hindered by several factors, making a multi-faceted approach necessary. Two factors contributing to injury are the irregular geometry of injured sites and the absence of tissue to hold potential nerve guides and drug therapies. Biocompatible hydrogels, injectable at room temperature, that rapidly solidify at physiological temperatures (37 °C) are beneficial materials that could hold nerve guidance channels in place and be loaded with therapeutic agents to aid wound healing. Our studies have shown that thermoreversible methylcellulose can be combined with agarose to create hydrogel blends that accommodate these properties. Three separate novel hydrogel blends were created by mixing methylcellulose with one of the three different agaroses. Gelation time tests show that the blends solidify at a faster rate than base methylcellulose at 37 °C. Rheological data showed that the elastic modulus of the hydrogel blends rapidly increases at 37 °C. Culturing experiments reveal that the morphology of dissociated dorsal root ganglion neurons was not altered when the hydrogels were placed onto the cells. The different blends were further assessed using dissolution tests, pore size evaluations using scanning electron microscopy and measuring the force required for injection. This research demonstrates that blends of agarose and methylcellulose solidify much more quickly than plain methylcellulose, while solidifying at physiological temperatures where agarose cannot. These hydrogel blends, which solidify at physiological temperatures naturally, do not require ultraviolet light or synthetic chemical cross linkers to facilitate solidification. Thus, these hydrogel blends have potential use in delivering therapeutics and holding scaffolding in place within the nervous system.

  13. Electrophoretic properties of the scrapie agent in agarose gels.

    OpenAIRE

    Prusiner, S B; Groth, D F; Bildstein, C; Masiarz, F R; McKinley, M P; Cochran, S P

    1980-01-01

    The molecular properties of the scrapie agent were investigated by subjecting partially purified preparations to electrophoresis on agarose gels. When electrophoresis was performed at room temperature in the presence of sodium dodecyl sulfate (NaDodSO4), most of the recoverable agent was found at the top of the gel, consistent with previous studies indicating aggregation of the agent upon exposure to elevated temperatures. In addition, less than 5% of the agent applied to the gel was found af...

  14. Separation of long RNA by agarose-formaldehyde gel electrophoresis

    OpenAIRE

    Mansour, Farrah H.; Pestov, Dimitri G.

    2013-01-01

    We describe a method to facilitate electrophoretic separation of high molecular weight RNA species, such as ribosomal RNAs and their precursors, on agarose-formaldehyde gels. Two alternative “pK-matched” buffer systems were substituted for the traditionally used MOPS-based conductive media. The key advantages include shortened run times, a five-fold reduction in formaldehyde concentration, a significantly improved resolution of long RNAs, and consistency in separation. The new procedure has a...

  15. Purification of coated vesicles by agarose gel electrophoresis

    OpenAIRE

    1981-01-01

    We have applied agarose gel electrophoresis as a novel step in the purification of clathrin-coated vesicles. Preparations of coated vesicles obtained by sedimentation velocity and isopycnic centrifugation are resolved into two distinct fractions upon electrophoresis. The slower migrating fraction contains smooth vesicles, whereas the faster contains only coated vesicles and empty clathrin coats. The faster mobility of the coated vesicles is primarily caused by the acidic nature of clathrin. C...

  16. Biomineral/Agarose Composite Gels Enhance Proliferation of Mesenchymal Stem Cells with Osteogenic Capability

    Directory of Open Access Journals (Sweden)

    Yoshika Suzawa

    2015-06-01

    Full Text Available Hydroxyapatite (HA or calcium carbonate (CaCO3 formed on an organic polymer of agarose gel is a biomaterial that can be used for bone tissue regeneration. However, in critical bone defects, the regeneration capability of these materials is limited. Mesenchymal stem cells (MSCs are multipotent cells that can differentiate into bone forming osteoblasts. In this study, we loaded MSCs on HA- or CaCO3-formed agarose gel and cultured them with dexamethasone, which triggers the osteogenic differentiation of MSCs. High alkaline phosphatase activity was detected on both the HA- and CaCO3-formed agarose gels; however, basal activity was only detected on bare agarose gel. Bone-specific osteocalcin content was detected on CaCO3-formed agarose gel on Day 14 of culture, and levels subsequently increased over time. Similar osteocalcin content was detected on HA-formed agarose on Day 21 and levels increased on Day 28. In contrast, only small amounts of osteocalcin were found on bare agarose gel. Consequently, osteogenic capability of MSCs was enhanced on CaCO3-formed agarose at an early stage, and both HA- and CaCO3-formed agarose gels well supported the capability at a later stage. Therefore, MSCs loaded on either HA- or CaCO3-formed agarose could potentially be employed for the repair of critical bone defects.

  17. Ion diffusion modelling of Fricke-agarose dosemeter gels

    International Nuclear Information System (INIS)

    In Fricke-agarose gels, an accurate determination of the spatial dose distribution is hindered by the diffusion of ferric ions. In this work, a model was developed to describe the diffusion process within gel samples of finite length and, thus, permit the reconstruction of the initial spatial distribution of the ferric ions. The temporal evolution of the ion concentration as a function of the initial concentration is derived by solving Fick's second law of diffusion in two dimensions with boundary reflections. The model was applied to magnetic resonance imaging data acquired at high spatial resolution (0.3 mm) and was found to describe accurately the observed diffusion effects. (authors)

  18. Orientation of the agarose gel matrix in pulsed electric fields.

    OpenAIRE

    Stellwagen, J; Stellwagen, N C

    1989-01-01

    The technique of transient electric birefringence was used to investigate the effect of pulsed electric fields on the orientation of the agarose gel matrix. Orientation of the gel was observed at all electric field strengths. Very slow, time-dependent effects were observed when pulses of 10-100 V/cm were applied to 1% gels for 0.5-2 seconds, indicating that domains of the matrix were being oriented by the electric field. The sign of the birefringence reversed when the direction of the applied...

  19. Immobilization of Candida cylindracea lipase on PVC, chitin and agarose

    Energy Technology Data Exchange (ETDEWEB)

    Chang, R.C.; Shaw, J.F.

    1987-01-01

    Candida cylindracea lipase was covalently coupled to PVC, chitin and agarose, which are abundant in Taiwan by several different methods. The agarose-dodecylene-diamine-glutaraldehyde (A-DDA-GA) system showed the highest relative loading enzyme activity, 118 mg soluble lipase per gram support. The chitosan-carbodiimide glutaraldehyde (CN-EDC-GA) systems immobilized 67 mg soluble lipase per gram support. The optimal pH of immobilized lipase was 8.5, which was one pH unit higher than that of soluble lipase. The optimal temperatures were in the range between 52-64/sup 0/C. The CN-EDC-GA system was the highest (64/sup 0/C), which was 27/sup 0/C higher than soluble lipase. The CH-EDC-GA system also had the best thermal stability (the half life at 55/sup 0/C was 29 h.) and operational stability at higher temperature (the half life at 40/sup 0/C was 495 h). However, the PVC-ethylenediamine-GA system appeared to have the best stability at lower temperature, the projected half life at 20/sup 0/C from Arrhenius plot was 31,802 h.

  20. Maintenance of biological activity of pertussis toxin radioiodinated while bound to fetuin-agarose.

    OpenAIRE

    Armstrong, G. D.; Peppler, M S

    1987-01-01

    We developed a method to produce radioiodinated pertussis toxin (PT) which was active in the goose erythrocyte agglutination and CHO cell assay systems. The procedure used fetuin coupled to agarose to prevent inactivation of the toxin during the iodination reaction. Analysis of the labeled PT by affinity chromatography on fetuin-agarose and wheat germ agglutinin-agarose and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that there were minimal amounts of labeled fetuin...

  1. Polyvinylpyrrolidone-Agarose Gel Electrophoresis Purification of Polymerase Chain Reaction-Amplifiable DNA from Soils

    OpenAIRE

    Young, Charles C.; Burghoff, Robert L.; Keim, Lois G.; Minak-Bernero, Vera; Lute, James R.; Hinton, Stephen M.

    1993-01-01

    This communication describes a modification of agarose gel electrophoresis to provide a rapid and simple method for the purification of polymerase chain reaction-amplifiable DNA from soil. This modification is to add polyvinylpyrrolidone to the agarose gel. The polyvinylpyrrolidone addition retards the electrophoretic mobility of denaturing phenolic compounds so that they do not comigrate with nucleic acids.

  2. Characterization of agarose as an encapsulation medium for particulate specimens for transmission electron microscopy.

    Science.gov (United States)

    Wood, J I; Klomparens, K L

    1993-07-01

    Agarose, agar, and gelatin were initially compared as encapsulation media for 3 structurally diverse particulate specimens: bacteria, yeast, and mitochondria. Agarose proved superior to both gelatin and agar for ease of handling and overall image quality (minimum background). All sample types exhibited high quality fixation and structural detail with no heat damage from the agarose medium. Based on this finding, we further characterized agarose encapsulation as affected by post-fixation, en bloc staining and resin type. Osmium tetroxide post-fixation, followed by en bloc uranyl acetate staining, could be performed without an increase in the electron density of the encapsulation medium. Agarose proved successful as an encapsulation medium regardless of the resin type or preparation protocol, thus providing flexibility in experimental design and excellent results over a range of variables. PMID:8358076

  3. Agarose and Polyacrylamide Gel Electrophoresis Methods for Molecular Mass Analysis of 5–500 kDa Hyaluronan

    OpenAIRE

    Bhilocha, Shardul; Amin, Ripal; Pandya, Monika; Yuan, Han; Tank, Mihir; LoBello, Jaclyn; Shytuhina, Anastasia; Wang, Wenlan; Wisniewski, Hans-Georg; De la Motte, Carol; Cowman, Mary K.

    2011-01-01

    Agarose and polyacrylamide gel electrophoresis systems for the molecular mass-dependent separation of hyaluronan (HA) in the size range of approximately 5–500 kDa have been investigated. For agarose-based systems, the suitability of different agarose types, agarose concentrations, and buffers systems were determined. Using chemoenzymatically synthesized HA standards of low polydispersity, the molecular mass range was determined for each gel composition, over which the relationship between HA ...

  4. Improving the culture of cucumber protoplasts by using an agarose-disc procedure

    International Nuclear Information System (INIS)

    A method is described for the isolation of protoplasts from cotyledons of cucumber (Cucumis sativus L.). After isolation, protoplasts were embedded in a mixture of agarose and Murashige and Skoog medium supplemented with 250 mg/L trypton, 2% sucrose, 5 μM naphthaleneacetic acid and 15 μM 2iP. The culture of the protoplasts was improved by the application of an agarose-disc culture procedure. The embedded protoplasts were plated in small (100 μL) droplets in Petri dishes to which, after gelling of the agarose, liquid medium was added. For comparison, protoplasts were also cultured according to the agarose-bead procedure. The plating efficiency ranged from 50 to 80% if the protoplasts were plated at high density (105 protoplasts per mL). In agarose-bead culture, divisions were induced at a lower rate. At lower densities the plating efficiency was dramatically decreased. Growth of microcalli was determined by homogenizing culture samples and measuring their density spectrophotometrically. The growth rate of the developing cell clusters was much higher in agarose-disc cultures as compared with bead-type cultures. It is concluded that for cucumber protoplasts the agarose-disc culture procedure provided optimal conditions for both the initiation of cell division and the growth of microcalli. (author)

  5. Synthesis of agarose-metal/semiconductor nanoparticles having superior bacteriocidal activity and their simple conversion to metal-carbon composites

    Indian Academy of Sciences (India)

    K K R Datta; B Srinivasan; H Balaram; M Eswaramoorthy

    2008-11-01

    Agarose, a naturally occurring biopolymer is used for the stabilization of metal, semiconductor nanoparticles. Ag and Cu nanoparticles stabilized in agarose matrix show excellent antibacterial activity against E. coli bacteria. The well dispersed metal nanoparticles within the agarose composite films can be readily converted to carbon-metal composites of catalytic importance.

  6. A simple immunoblotting method after separation of proteins in agarose gel

    DEFF Research Database (Denmark)

    Koch, C; Skjødt, K; Laursen, I

    A simple and sensitive method for immunoblotting of proteins after separation in agarose gels is described. It involves transfer of proteins onto nitrocellulose paper simply by diffusion through pressure, a transfer which only takes about 10 min. By this method we have demonstrated the existence of...... multiple molecular forms of the complement factors C3 and factor B in serum from 2 species, man and chicken, after electrophoretic separation in agarose. We have also demonstrated the usefulness of the method for determining the isoelectric point of proteins after isoelectric focusing in agarose....

  7. Investigation of physical-chemical properties of agarose hydrogels with embedded emulsions.

    Science.gov (United States)

    Komarova, Galina A; Starodubtsev, Sergey G; Khokhlov, Alexei R

    2009-11-12

    Composite agarose hydrogels with embedded tetradecane emulsions stabilized by cetylpyridinium chloride were studied. The absorption efficiency of 4-nitrophenyl ethers of carbonic acids by the composite agarose gels increases with the length of the hydrocarbon tail of the ester. The diffusion rate of amphiphilic substances in the composite gels was demonstrated to be much less that than in the standard agarose gels. The reaction kinetics between the esters and dodecylmercaptan dissolved in tetradecane droplets of composite hydrogel was studied. In the region of physiological pH, the reactivity of SH groups embedded in the composite agarose gel in the reaction with the esters is significantly higher than that in a homogeneous solution. Hydrogels with embedded emulsion droplets are of considerable practical importance as drug delivery systems, microreactors, and absorbers. Composite gels filled with emulsions incorporating lipophilic mercaptanes are effective absorbers of heavy metal ions. PMID:19835385

  8. Searching for the best agarose candidate from genusGracilaria,Eucheuma,Gelidium and local brands

    Institute of Scientific and Technical Information of China (English)

    Ferry Efendi; Retno Handajani; Nursalam Nursalam

    2015-01-01

    Objective:To explore the potential of local agar of genusGracilaria,Eucheuma,Gelidium and local brandsas an alternative for imported agarose forDNA electrophoresis, and to examine their ability related to separation and migration ofDNA fragments inDNA electrophoresis. Methods:Their performance at various concentrations were compared via an experimental study with a specific brand of imported commercial agarose used in molecular biology research. The measured variables were separation and migration during electrophoresis of a DNA fragment. Results: The local agar genusGracilaria gigas,Gelidium, brand "B" and brand "S" could separateDNA fragments at a concentration between 1% and 2%, with an optimum concentration of 2% w/v, as good as a specific brand of imported commercial agarose. Conclusions:Their performance were very close to that of commercial agarose and can still be improved by further agar purification as well as by pH and sulfur control.

  9. Agarose gel shift assay reveals that calreticulin favors substrates with a quaternary structure in solution

    DEFF Research Database (Denmark)

    Boelt, Sanne Grundvad; Houen, Gunnar; Højrup, Peter

    2015-01-01

    Here we present an agarose gel shift assay that, in contrast to other electrophoresis approaches, is loaded in the center of the gel. This allows proteins to migrate in either direction according to their isoelectric points. Therefore, the presented assay enables a direct visualization, separation......-sheets in their secondary structure. It is also demonstrated that the agarose gel shift assay is useful in the study of other protein interactions and can be used as an alternative method to native polyacrylamide gel electrophoresis....

  10. Subpopulations of liver coated vesicles resolved by preparative agarose gel electrophoresis

    OpenAIRE

    1986-01-01

    Rat liver clathrin coated vesicles (CVs) were separated into several distinct subpopulations using non-sieving concentrations of agarose, which allowed the separation of species differing primarily in surface charge. Using preparative agarose electrophoresis (Kedersha, N. L., and L. H. Rome, 1986, Anal. Biochem., in press), the CVs were recovered and analyzed for differences in morphology, coat protein composition, and stripped vesicle protein composition. Coat proteins from different populat...

  11. Use of agarose gel electrophoresis of plasmid deoxyribonucleic acid to fingerprint gram-negative bacilli.

    OpenAIRE

    Schaberg, D.R.; Tompkins, L S; Falkow, S

    1981-01-01

    Agarose gel electrophoresis of the plasmid deoxyribonucleic acids from 60 gram-negative bacilli recovered during investigations of nosocomial epidemics was used to fingerprint the strains. This method was as specific at differentiating bacterial strains as more conventional phenotyping methods. In all cases, plasmid band fingerprints of epidermic strains isolates were identical whereas coisolate plasmid deoxyribonucleic acid patterns were different. Agarose gel electrophoresis of plasmid deox...

  12. Ultra-deep desulfurization via reactive adsorption on peroxophosphomolybdate/agarose hybrids.

    Science.gov (United States)

    Xu, Jian; Li, Huacheng; Wang, Shengtian; Luo, Fang; Liu, Yunyu; Wang, Xiaohong; Jiang, Zijiang

    2014-09-01

    A catalyst system composed of peroxophosphomolybdates as catalytic center and agarose as matrix material had been designed. The [C16H33N(CH3)3]3[PO4{MoO(O2)2}4]/agarose (C16PMo(O2)2/agarose) hybrid was found to be active for oxidation desulfurization (ODS) of dibenzothiophene (DBT) or real fuel into corresponding sulfone by H2O2 as an oxidant, while the sulfur content could be reduced to 5ppm. The higher activity comes from its components including [PO4{MoO(O2)2}4] catalytic sites, the hydrophobic quaternary ammonium cation affinity to low polarity substrates, and agarose matrix affinity to H2O2 and sulfone. During the oxidative reaction, the mass transfer resistance between H2O2 and organic sulfurs could be decreased and the reaction rate could increase by the assistance of agarose and hydrophobic tails of [C16H33N(CH3)3]3[PO4{MoO(O2)2}4]. Meanwhile, the oxidative products could be adsorbed by agarose matrix to give clean fuel avoiding the post-treatment. In addition, the hybrid was easily regenerated to be reused. PMID:24997975

  13. A microfluidic device for on-chip agarose microbead generation with ultralow reagent consumption.

    Science.gov (United States)

    Desbois, Linda; Padirac, Adrien; Kaneda, Shohei; Genot, Anthony J; Rondelez, Yannick; Hober, Didier; Collard, Dominique; Fujii, Teruo

    2012-01-01

    Water-in-oil microdroplets offer microreactors for compartmentalized biochemical reactions with high throughput. Recently, the combination with a sol-gel switch ability, using agarose-in-oil microdroplets, has increased the range of possible applications, allowing for example the capture of amplicons in the gel phase for the preservation of monoclonality during a PCR reaction. Here, we report a new method for generating such agarose-in-oil microdroplets on a microfluidic device, with minimized inlet dead volume, on-chip cooling, and in situ monitoring of biochemical reactions within the gelified microbeads. We used a flow-focusing microchannel network and successfully generated agarose microdroplets at room temperature using the "push-pull" method. This method consists in pushing the oil continuous phase only, while suction is applied to the device outlet. The agarose phase present at the inlet is thus aspirated in the device, and segmented in microdroplets. The cooling system consists of two copper wires embedded in the microfluidic device. The transition from agarose microdroplets to microbeads provides additional stability and facilitated manipulation. We demonstrate the potential of this method by performing on-chip a temperature-triggered DNA isothermal amplification in agarose microbeads. Our device thus provides a new way to generate microbeads with high throughput and no dead volume for biochemical applications. PMID:24106525

  14. Two methods that facilitate autoradiography of small 32P-labeled DNA fragments following electrophoresis in agarose gels

    International Nuclear Information System (INIS)

    Two methods which permit detection by autoradiography of small 32P-labeled DNA fragments resolved by agarose gel electrophoresis are described. Agarose gel electrophoresis poses problems for autoradiography as (i) the gels are normally too thick to allow autoradiography without being dried first, and (ii) fragments of DNA of 1000 bp or less in length are readily lost during drying. In this study DNA fragments as small as 121 bp have been retained in agarose gels upon drying. This has been achieved by either (i) first fixing the DNA with the cationic detergent cetyltrimethylammonium bromide, or (ii) drying the agarose gels onto Zeta-Probe charge-modified membranes

  15. Plasticizing effect of choline chloride/urea eutectic-based ionic liquid on physicochemical properties of agarose films

    Directory of Open Access Journals (Sweden)

    Ahmad Adlie Shamsuri

    2012-11-01

    Full Text Available Agarose films were formed with the addition of 30 to 70 wt% choline chloride/urea eutectic-based ionic liquid (ChCl/Urea. The ChCl/Urea was prepared through complexation at a 1:2 mole ratio. The films were prepared by dissolving ChCl/Urea in distilled water followed by dispersion of the agarose at 95 °C. The solution was gelled at room temperature, and the formed gel was dried in an oven overnight at 70 °C. Mechanical testing indicated that the agarose film containing 60 wt% ChCl/Urea had higher tensile extension and tensile strain at break compared to the pristine agarose film. The addition of ChCl/Urea also reduced the glass transition temperature (Tg of agarose films. Cross-section SEM images of the agarose films showed that surface roughness disappeared with the incorporation of ChCl/Urea. FTIR spectra confirmed the presence of intermolecular hydrogen bonding between agarose and ChCl/Urea. XRD patterns demonstrated that an amorphous phase was obtained when ChCl/Urea was added. Agarose films containing more ChCl/Urea exhibited higher transparency, as measured by a UV-Vis spectrometer. In summary, the physicochemical properties of agarose films were evidently affected by the incorporation of the ChCl/Urea as a plasticizing agent.

  16. Bridge DNA amplification of cancer-associated genes on cross-linked agarose microbeads

    International Nuclear Information System (INIS)

    A cross-linked agarose substrate was studied as a 3D support for bridge solid-phase DNA amplification (SPA). In this kind of SPA, primers are immobilized on agarose beads. Flow cell studies of SPA in real-time experiments showed that the amplification efficiency is strongly affected by (a) the presence of a linker between the primer and substrate, and (b) by the loading with primers. In fact, a high loading density may compromise SPA. The analysis of real time SPA curves using geometric growth model highlighted the advantage of 3D agarose support over the flat surfaces. The potential of bridge 3D SPA in DNA diagnostics was successfully demonstrated by on-chip analysis of mutations of the cancer-associated genes BRCA1/2 and CHEK2. (author)

  17. Ag-nanoparticle fractionation by low melting point agarose gel electrophoresis

    International Nuclear Information System (INIS)

    The separation of surface-enhanced raman scattering (SERS)-active Ag-multi-nanoparticle (NP) assemblies by low melting point agarose gel electrophoresis was accomplished here by controlling surface charge using NP capping agents, and the pore size of agarose gel matrix. Detailed transmission electron microscopy analysis of excised gel fractions showed dimers and small clusters to have the greatest SERS activity and a mobility in between the monomers and large aggregates. This strategy enables one to: (1) stabilize small multispherical Ag clusters against further aggregation during purification; (2) fractionate and recover spherical assemblies by nuclearity; and (3) analyze SERS-enhancements for each fraction to optimize purification conditions.

  18. Comparison of viability of adipose-derived Mesenchymal stem cells on agarose and fibrin glue scaffolds

    Directory of Open Access Journals (Sweden)

    Farzaneh Tafvizi

    2015-06-01

    Full Text Available Background & aim: Utilizing tissue engineering techniques and designing similar structures of the damaged tissues require the use of tools such as scaffolds, cells, and bioactive molecules in vitro. Meanwhile, appropriate cell cultures with the ability to divide and differentiate on the natural scaffolds lacking features like immunogenicity and tumorgenesis is particularly important. Adipose tissue has attracted researchers’ attention due to its abundance of mesenchymal stem cells and its availability through a liposuction. The purpose of the present study was to investigate the reproducibility and viability of the adipose-derived stem cells on natural scaffolds of fibrin glue and agarose. Methods: In the present experimental study, the isolation and identification of the mesenchymal stem cells was performed on tissue obtained from liposuction. The tissues were extensively washed with PBS and were digested with collagenase I, then the mesenchymal stem cells were isolated. The cells were cultured in RPMI medium supplemented with antibiotic. Subsequently, the expression of cell surface markers including CD34, CD44, CD90, and CD105 were analyzed by flow cytometry to confirm the mesenchymal cells. After preparing fibrin glue and agarose scaffolds, the viability and proliferation of the adipose tissue-derived mesenchymal stem cells were examined at the period of 24, 48, and 72 hours by MTT and ELISA assays. The obtained results were analyzed by SPSS ver.19. Results: The results of adipose tissue-derived mesenchymal stem cells culture on the fibrin glue and agarose scaffolds indicated that cell viability on fibrin glue and agarose scaffold were 68.22% and 89.75% in 24 hrs, 64.04% and 66.97% in 48 hours, 222.87% and 1089.68% in 72 hours respectively. Significant proliferation and viability cells on a synthesized agarose scaffold were seen compared to the fibrin glue scaffold after 72 hrs. The viability of the cells significantly increased on the

  19. Separation of 1–23-kb complementary DNA strands by urea–agarose gel electrophoresis

    OpenAIRE

    Hegedüs, Éva; Kókai, Endre; Kotlyar, Alexander; Dombrádi, Viktor; Szabó, Gábor

    2009-01-01

    Double-stranded (ds), as well as denatured, single-stranded (ss) DNA samples can be analyzed on urea–agarose gels. Here we report that after denaturation by heat in the presence of 8 M urea, the two strands of the same ds DNA fragment of ∼1–20-kb size migrate differently in 1 M urea containing agarose gels. The two strands are readily distinguished on Southern blots by ss-specific probes. The different migration of the two strands could be attributed to their different, base composition-depen...

  20. A new polymer gel dosimeter composed of methacrylic acid, agarose gel and THPC with gelatin

    International Nuclear Information System (INIS)

    In this paper, a new type of methacrylic acid based gel dosimeter is presented. This gel contains both agarose and gelatin with deferent roles respectively. The agarose conducts itself as a gelling agent, while the gelatin relates to the graft reaction of methacrylic acid. This new type of gel excels in the long-term stability of R2 after irradiation. The characteristics of this gel were studied by the measurements of R2 with MRI and the direct measurements of temperature in the gel during the irradiation.

  1. Dye-sensitized solar cells with ionic gel electrolytes prepared from imidazolium salts and agarose

    International Nuclear Information System (INIS)

    New ionic gel electrolytes, semi-solid state electrolytes comprised of ionic liquid and gelator were investigated in order to improve the durability of dye-sensitized solar cells (DSCs). The ionic gels were prepared from agarose, natural polysaccharide, and 1-alkyl-3-methyl-imidazolium salts. The gels showed sufficient mechanical strength even though a very small amount of agarose was added (1.0-1.5 wt%). The photon to electron conversion efficiency of the DSCs containing ionic gel electrolyte was 2.93% under simulated sunlight (air mass 1.5) with a light intensity of 100 mW cm-2. (authors)

  2. Ag-nanoparticle fractionation by low melting point agarose gel electrophoresis

    Energy Technology Data Exchange (ETDEWEB)

    Guarrotxena, Nekane, E-mail: nekane@ictp.csic.es [Consejo Superior de Investigaciones Cientificas (CSIC), Instituto de Ciencia y Tecnologia de Polimeros (ICTP) (Spain); Braun, Gary [University of California, Santa Barbara, Department of Chemistry and Biochemistry (United States)

    2012-10-15

    The separation of surface-enhanced raman scattering (SERS)-active Ag-multi-nanoparticle (NP) assemblies by low melting point agarose gel electrophoresis was accomplished here by controlling surface charge using NP capping agents, and the pore size of agarose gel matrix. Detailed transmission electron microscopy analysis of excised gel fractions showed dimers and small clusters to have the greatest SERS activity and a mobility in between the monomers and large aggregates. This strategy enables one to: (1) stabilize small multispherical Ag clusters against further aggregation during purification; (2) fractionate and recover spherical assemblies by nuclearity; and (3) analyze SERS-enhancements for each fraction to optimize purification conditions.

  3. Effect of alternation of agar and agarose on the green plant differentiation frequency of calli from wild rice

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    @@ In vitro culture of wild rice was difficult. Recently, we found that high agarose concentration of different media could improve the green plant differentiation frequency of calli from wild rice. We tested the effectiveness of alternation of agar and agarose in different media.

  4. In situ DNA extraction from bacterial spores distributed on agarose gel using atmospheric pressure cold plasma

    Energy Technology Data Exchange (ETDEWEB)

    Yasuda, H.; Tanino, Y.; Takashima, K.; Mizuno, A. [Toyohashi Univ. of Technology, Toyohashi (Japan). Dept. of Ecological Engineering

    2010-07-01

    This paper presented a newly proposed systematic method for high speed counting of airborne bioparticles (BPs) that can be used in different indoor environments such as hospitals, pharmaceutical or food processing companies. The method involves plasma lysis of the BPs and detection of DNA cells. In this study, BPs were distributed on the surface of agarose gel to destroy cell walls using dielectric barrier discharge (DBD). DNA cells were detected by electro-blotting onto a membrane filter. The use of DBD enabled the extraction of the chromosomal DNA in situ from many types of cells without resorting to cell wall digesting enzymes. Bacillus subtilis spores were used because they are highly resistant to harsh physical and chemical treatments. The Bacillus subtilis spores were placed at low density on an agarose gel. The application of DBD destroyed the spores, but the chromosome DNA remained without extensive degradation. The electrophoresis of the DNA through the agarose gel to a membrane filter was examined to separate the DNA from the other substances in the lysates. The study showed that sufficient DNA was transferred to the filter through the agarose gel layer without scattering, almost keeping the original shape of the bacteria. Nucleic acid biomarkers will be used for bacterial identification of the DNA immobilized filter.

  5. Molecular analysis of chondrocytes cultured in agarose in response to dynamic compression

    Directory of Open Access Journals (Sweden)

    Mallein-Gerin Frédéric

    2008-09-01

    Full Text Available Abstract Background Articular cartilage is exposed to high mechanical loads under normal physiological conditions and articular chondrocytes regulate the composition of cartilaginous matrix, in response to mechanical signals. However, the intracellular pathways involved in mechanotransduction are still being defined. Using the well-characterized chondrocyte/agarose model system and dynamic compression, we report protocols for preparing and characterizing constructs of murine chondrocytes and agarose, and analyzing the effect of compression on steady-state level of mRNA by RT-PCR, gene transcription by gene reporter assay, and phosphorylation state of signalling molecules by Western-blotting. The mouse model is of particular interest because of the availability of a large choice of bio-molecular tools suitable to study it, as well as genetically modified mice. Results Chondrocytes cultured in agarose for one week were surrounded by a newly synthesized pericellular matrix, as revealed by immunohistochemistry prior to compression experiments. This observation indicates that this model system is suitable to study the role of matrix molecules and trans-membrane receptors in cellular responsiveness to mechanical stress. The chondrocyte/agarose constructs were then submitted to dynamic compression with FX-4000C™ Flexercell® Compression Plus™ System (Flexcell. After clearing proteins off agarose, Western-blotting analysis showed transient activation of Mitogen-activated protein kinases (MAPK in response to dynamic compression. After assessment by capillary electrophoresis of the quality of RNA extracted from agarose, steady-state levels of mRNA expression was measured by real time PCR. We observed an up-regulation of cFos and cJun mRNA levels as a response to compression, in accordance with the mechanosensitive character observed for these two genes in other studies using cartilage explants submitted to compression. To explore further the

  6. Pulsatile dynamic stiffness of cartilage-like materials and use of agarose gels to validate mechanical methods and models.

    Science.gov (United States)

    Scandiucci de Freitas, P; Wirz, D; Stolz, M; Göpfert, B; Friederich, N-F; Daniels, A U

    2006-08-01

    Stiffness is a fundamental indicator of the functional state of articular cartilage. Reported test modes include compressive incremental strain to determine the equilibrium modulus, and sinusoidal strain to determine the dynamic modulus and stress/strain loss angle. Here, initial development is described for a method recognizing that gait is pulsatile. Agarose gels have been used by others for validation or comparison of mechanical test methods and models for cartilage and proteoglycan aggregate. Accordingly, gels ranging from 0.5 to 20% agarose were prepared. Pulsatile stiffness in both indentation and unconfined compression were closely reproducible. Stiffness as a function of agarose concentration rose exponentially, as found using other methods. Indentation stiffness was higher than for unconfined compression and ranged from approximately 2.0 kPa for 0.5% gel to approximately 3,800 kPa for 20% gel. Pulsatile dynamic stiffness appears to be a useful method, although further development is needed. Agarose gel stiffness values obtained by other methods were reviewed for comparison. Unfortunately, reported values for a given agarose concentration ranged widely (e.g. fourfold) even when test methods were similar. Causes appear to include differences in molecular weight and gel preparation time-temperature regimens. Also, agarose is hygroscopic, leading to unintended variations in gel composition. Agarose gels are problematic materials for validation or comparison of cartilage mechanical test methods and models. PMID:16470817

  7. Agarose functionalization: Synthesis of PEG-agarose amino acid nano-conjugate - its structural ramifications and interactions with BSA in a varying pH regime.

    Science.gov (United States)

    Chudasama, Nishith A; Prasad, Kamalesh; Siddhanta, Arup Kumar

    2016-10-20

    In a rapid one-step method protein-mimicking large agarose amino acid framework (AAE; GPC 156.7kDa) was conjugated with polyethylene glycol (PEG 9kDa) affording nano-sized PEGylated amphoteric agarose (PEG-AAE; amino, carboxyl and ester groups [overall degree of substitution (DS) 0.91]. The PEG groups were at the residual free carboxylic acid groups of succinate half-ester moiety at C-6 positions of the 1, 3 β-d-galactopyranose moieties of AAE. This new nano-sized PEG-AAE performed like a giant protein conjugate (GPC 331.2kDa) and exhibited pH-responsive interconversion between the triple helix and single-stranded random structures (optical rotatory dispersion) presenting a mixed solubility pattern like random coil (soluble), helical (soluble) and aggregate (precipitation) formations. Circular dichroism studies showed its pH-dependent complexation and decomplexation with bovine serum albumin (BSA). Such pH-responsive PEG-conjugate may be of pronounced therapeutic potential in the area of pharmacology as well as in sensing applications. PMID:27474620

  8. High-Yield Separation of Metallic and Semiconducting Single-Wall Carbon Nanotubes by Agarose Gel Electrophoresis

    Science.gov (United States)

    Tanaka, Takeshi; Jin, Hehua; Miyata, Yasumitsu; Kataura, Hiromichi

    2008-11-01

    We have developed a novel separation method of metallic and semiconducting single-wall carbon nanotubes (SWCNTs) using agarose gel electrophoresis. When the SWCNTs were isolated with sodium dodecyl sulfate (SDS) and embedded in agarose gel, only the metallic SWCNTs separated from the starting gel by an electric field. After 20 min, almost all SWCNTs applied to gel electrophoresis were separated into two fractions, containing ˜95% semiconducting and ˜70% metallic nanotubes. The difference in the response to the electric field between metallic and semiconducting SWCNTs can be explained by the higher affinity of semiconducting SWCNTs to agarose than to SDS.

  9. Preparation of berbamine loaded chitosan-agarose microspheres and in vitro release study

    Directory of Open Access Journals (Sweden)

    Zhang Hu

    2012-01-01

    Full Text Available Berbamine loaded chitosan-agarose microspheres were prepared using a water-in-oil emulsion technique. Optimum preparing parameters were determined by orthogonal experiments as follows: ratio of berbamine to chitosan (w/w is 1:10; percentage of emulsifier (span 80, v/v is 6%; volume of glutaraldehyde is 2 mL; and reaction temperature is 70 ºC. Under these optimal conditions, the encapsulation efficiency and loading capacity of microspheres are 84.57% and 8.44%, respectively. The swelling tests showed that the microspheres possessed higher swelling ratio at pH 7.4 than at pH 1.2. FTIR indicated that berbamine had been successfully loaded in the chitosan-agarose microspheres by physical entrapment. In vitro release studies showed that berbamine was released from microspheres in a significantly sustained fashion.

  10. Recovery of DNA from Agarose Gel with Home-made Silica Milk

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    An usefulness of silica milk made with waste ultraviolet light tube for recovery of DNA fragment from agarose gel was represented. The glass milk is a water suspension of 50% fine silica powder prepared by grinding the crushed waste ultraviolet light tube with a porcelain mortar.It was showed that one microliter of the glass milk could bind more than 1 μg of DNA fragment,and DNA fragment in length from 125 bp to 23 kb could be efficiently recovered from agarose gel. The bound DNA could be eluted from the particle of SiO2 in the glass milk with a yield of about 60%-80%.The eluted DNA could be used in all manipulations in molecular cloning.

  11. Preparation and structural characterization of O-acetyl agarose with low degree of substitution

    Directory of Open Access Journals (Sweden)

    Rosangela B. Garcia

    2000-09-01

    Full Text Available Among the biodegradable polymers, the polysaccharides have been found to be promising carriers for bioactive molecules. From a general standpoint, they present several reactive groups, such as hydroxyl, carboxyl and amine, that can be modified in a number of ways, giving rise to suitable devices for controlled release. In this paper, agarose was submitted to O-acetylation reactions under heterogeneous conditions, using acetic anhydride and pyridine, aiming to observe the effect of acetyl groups on the agarose properties. The products were characterized by Infrared and ¹H NMR spectroscopies. In the range of average acetylation degrees (DA 0.07-0.48, the polymers presented partial solubility in boiling water and in common organic solvents. The ¹H NMR spectra presented evidences of non-homogeneous acetyl group distribution along the chains, as concluded from the solubility of only one of the fractions with DA<0.09, in boiling water .

  12. Kinetic model for whey protein hydrolysis by alcalase multipoint-immobilized on agarose gel particles

    OpenAIRE

    Sousa Jr R.; Lopes G. P.; Tardioli P. W.; Giordano R.L.C.; Almeida P. I. F.; Giordano R. C.

    2004-01-01

    Partial hydrolysis of whey proteins by enzymes immobilized on an inert support can either change or evidence functional properties of the produced peptides, thereby increasing their applications. The hydrolysis of sweet cheese whey proteins by alcalase, which is multipoint-immobilized on agarose gel, is studied here. A Michaelis-Menten model that takes into account competitive inhibition by the product was fitted to experimental data. The influence of pH on the kinetic parameters in the range...

  13. Plasmid DNA replication and topology as visualized by two-dimensional agarose gel electrophoresis

    OpenAIRE

    Schvartzman, Jorge Bernardo; Martínez-Robles, María Luisa; Krimer, Dora B.

    2010-01-01

    During the last 20 years, two-dimensional agarose gel electrophoresis combined with other techniques such as Polymerase Chain Reaction, helicase assay and electron microscopy, helped to characterize plasmid DNA replication and topology. Here we describe some of the most important findings that were made using this method including the characterization of uni-directional replication, replication origin interference, DNA breakage at the forks, replication fork blockage, replication knotting, re...

  14. Trapping of megabase-sized DNA molecules during agarose gel electrophoresis

    OpenAIRE

    Gurrieri, Sergio; Smith, Steven B.; Bustamante, Carlos

    1999-01-01

    Megabase DNA molecules become trapped in agarose gels during electrophoresis if the electric field exceeds a few volts per cm. Fluorescence microscopy reveals that these molecules invariably arrest in U-shaped conformations. The field-vs.-size dependence for trapping indicates that a critical molecular tension is required for trapping. The size of unligated λ-ladders, sheared during gel electrophoresis at a given field, coincides with the size of molecules trapped at that field, suggesting th...

  15. An evaluation of cerebrospinal fluid oligoclonal banding confirmed by immunofixation on agarose gel.

    OpenAIRE

    George, P M; Lorier, M A; Donaldson, I M

    1983-01-01

    The cerebrospinal fluid (CSF) from 115 consecutive patients undergoing diagnostic lumbar puncture or myelography was examined to determine the usefulness of immunofixation, following agarose gel electrophoresis, in the detection of oligoclonal IgG. All electrophoretic patterns were evaluated with and without immunofixation, and the interpretation of 9% of specimens was altered by immunofixation. The demonstration of oligoclonal IgG was shown to be more reliable in the diagnosis of multiple sc...

  16. Mesoscopic gel at low agarose concentration in water: a dynamic light scattering study.

    OpenAIRE

    Bulone, D; San Biagio, P L

    1995-01-01

    Previous work in our laboratory has shown that at very low agarose concentration in water gelation still occurs within mutually disconnected, high concentration regions generated by spinodal demixing. The freely diffusing particles obtained in these conditions are studied in the present work by depolarized dynamic light scattering and probe diffusion experiments. These particles are found to behave as large (in fact, mesoscopic) polymer fibers entangled in a continuously rearranged mesh with ...

  17. A disposable bio-nano-chip using agarose beads for high performance immunoassays

    OpenAIRE

    Du, Nan; Chou, Jie; Kulla, Eliona; Floriano, Pierre N.; Christodoulides, Nicolaos; McDevitt, John T.

    2011-01-01

    This article reports on the fabrication of a disposable bio-nano-chip (BNC), a microfluidic device composed of polydimethylsiloxane (PDMS) and thiolene-based optical epoxy which is both cost-effective and suitable for high performance immunoassays. A novel room temperature (RT) bonding technique was utilized so as to achieve irreversible covalent bonding between PDMS and thiolene-based epoxy layers, while at the same time being compatible with the insertion of agarose bead sensors, selectivel...

  18. Retention of gene expression in porcine islets after agarose encapsulation and long-term culture.

    Science.gov (United States)

    Dumpala, Pradeep R; Holdcraft, Robert W; Martis, Prithy C; Laramore, Melissa A; Parker, Thomas S; Levine, Daniel M; Smith, Barry H; Gazda, Lawrence S

    2016-08-01

    Agarose encapsulation of porcine islets allows extended in vitro culture, providing ample time to determine the functional capacity of the islets and conduct comprehensive microbiological safety testing prior to implantation as a treatment for type 1 diabetes mellitus. However, the effect that agarose encapsulation and long-term culture may have on porcine islet gene expression is unknown. The aim of the present study was to compare the transcriptome of encapsulated porcine islets following long-term in vitro culture against free islets cultured overnight. Global gene expression analysis revealed no significant change in the expression of 98.47% of genes. This indicates that the gene expression profile of free islets is highly conserved following encapsulation and long-term culture. Importantly, the expression levels of genes that code for critical hormones secreted by islets (insulin, glucagon, and somatostatin) as well as transcripts encoding proteins involved in their packaging and secretion are unchanged. While a small number of genes known to play roles in the insulin secretion and insulin signaling pathways are differentially expressed, our results show that overall gene expression is retained following islet isolation, agarose encapsulation, and long-term culture. PMID:27261433

  19. Preparation of uniform-sized agarose beads by microporous membrane emulsification technique.

    Science.gov (United States)

    Zhou, Qing-Zhu; Wang, Lian-Yan; Ma, Guang-Hui; Su, Zhi-Guo

    2007-07-01

    Uniform-sized agarose beads were prepared by membrane emulsification technique in this study. Agarose was dissolved in boiling water (containing 0.9% sodium chloride) and used as water phase. A mixture of liquid paraffin and petroleum ether containing 4 wt% of hexaglycerin penta ester (PO-500) emulsifier was used as oil phase. At 55 degrees C, the water phase permeated through uniform pores of microporous membrane into the oil phase by a pressure of nitrogen gas to form uniform W/O emulsion. Then the emulsion was cooled down to room temperature under gentle agitation to form gel beads. The effect of oil phase, emulsifier, especially temperature on the uniformity of the beads were investigated and interpreted from interfacial tension between water phase and oil phase. Under optimized condition, the coefficient variation (C.V.) showing the size distribution of the beads was under 15%. This was the first report to prepare uniform agarose beads by membrane emulsification, and to investigate the effect of temperature on the size distribution of the droplets and beads. The beads with different size can be prepared by using membranes with different pore size, and the result showed that there was a linear relationship between the average diameter of beads and pore size of the membranes; beads with diameter from 15 to 60 microm were able to obtain in this study. PMID:17362974

  20. Fenugreek hydrogel–agarose composite entrapped gold nanoparticles for acetylcholinesterase based biosensor for carbamates detection

    International Nuclear Information System (INIS)

    A biosensor was fabricated to detect pesticides in food samples. Acetylcholinesterase was immobilized in a novel fenugreek hydrogel–agarose matrix with gold nanoparticles. Transparent thin films with superior mechanical strength and stability were obtained with 2% fenugreek hydrogel and 2% agarose. Immobilization of acetylcholinesterase on the membrane resulted in high enzyme retention efficiency (92%) and a significantly prolonged shelf life of the enzyme (half-life, 55 days). Transmission electron microscopy revealed that, gold nanoparticles (10–20 nm in diameter) were uniformly dispersed in the fenugreek hydrogel–agarose–acetylcholinesterase membrane. This immobilized enzyme-gold nanoparticle dip-strip system detected various carbamates, including carbofuran, oxamyl, methomyl, and carbaryl, with limits of detection of 2, 21, 113, and 236 nM (S/N = 3), respectively. Furthermore, the fabricated biosensor exhibited good testing capabilities when used to detect carbamates added to various fruit and vegetable samples. - Highlights: • Acetylcholinesterase (AChE) dip-strip biosensor fabricated to detect carbamates. • AChE entrapped in fenugreek hydrogel–agarose matrix with gold nanoparticles (GNPs). • High enzyme retention efficiency (92%) and shelf life (half-life, 55 days). • Detection limits of carbofuran, oxamyl and methomyl: 2, 21 and 113 nM. • The biosensor had good testing capabilities to detect carbamates in food samples

  1. Fenugreek hydrogel–agarose composite entrapped gold nanoparticles for acetylcholinesterase based biosensor for carbamates detection

    Energy Technology Data Exchange (ETDEWEB)

    Kestwal, Rakesh Mohan; Bagal-Kestwal, Dipali; Chiang, Been-Huang, E-mail: bhchiang@ntu.edu.tw

    2015-07-30

    A biosensor was fabricated to detect pesticides in food samples. Acetylcholinesterase was immobilized in a novel fenugreek hydrogel–agarose matrix with gold nanoparticles. Transparent thin films with superior mechanical strength and stability were obtained with 2% fenugreek hydrogel and 2% agarose. Immobilization of acetylcholinesterase on the membrane resulted in high enzyme retention efficiency (92%) and a significantly prolonged shelf life of the enzyme (half-life, 55 days). Transmission electron microscopy revealed that, gold nanoparticles (10–20 nm in diameter) were uniformly dispersed in the fenugreek hydrogel–agarose–acetylcholinesterase membrane. This immobilized enzyme-gold nanoparticle dip-strip system detected various carbamates, including carbofuran, oxamyl, methomyl, and carbaryl, with limits of detection of 2, 21, 113, and 236 nM (S/N = 3), respectively. Furthermore, the fabricated biosensor exhibited good testing capabilities when used to detect carbamates added to various fruit and vegetable samples. - Highlights: • Acetylcholinesterase (AChE) dip-strip biosensor fabricated to detect carbamates. • AChE entrapped in fenugreek hydrogel–agarose matrix with gold nanoparticles (GNPs). • High enzyme retention efficiency (92%) and shelf life (half-life, 55 days). • Detection limits of carbofuran, oxamyl and methomyl: 2, 21 and 113 nM. • The biosensor had good testing capabilities to detect carbamates in food samples.

  2. High throughput generation and trapping of individual agarose microgel using microfluidic approach

    KAUST Repository

    Shi, Yang

    2013-02-28

    Microgel is a kind of biocompatible polymeric material, which has been widely used as micro-carriers in materials synthesis, drug delivery and cell biology applications. However, high-throughput generation of individual microgel for on-site analysis in a microdevice still remains a challenge. Here, we presented a simple and stable droplet microfluidic system to realize high-throughput generation and trapping of individual agarose microgels based on the synergetic effect of surface tension and hydrodynamic forces in microchannels and used it for 3-D cell culture in real-time. The established system was mainly composed of droplet generators with flow focusing T-junction and a series of array individual trap structures. The whole process including the independent agarose microgel formation, immobilization in trapping array and gelation in situ via temperature cooling could be realized on the integrated microdevice completely. The performance of this system was demonstrated by successfully encapsulating and culturing adenoid cystic carcinoma (ACCM) cells in the gelated agarose microgels. This established approach is simple, easy to operate, which can not only generate the micro-carriers with different components in parallel, but also monitor the cell behavior in 3D matrix in real-time. It can also be extended for applications in the area of material synthesis and tissue engineering. © 2013 Springer-Verlag Berlin Heidelberg.

  3. A disposable bio-nano-chip using agarose beads for high performance immunoassays.

    Science.gov (United States)

    Du, Nan; Chou, Jie; Kulla, Eliona; Floriano, Pierre N; Christodoulides, Nicolaos; McDevitt, John T

    2011-10-15

    This article reports on the fabrication of a disposable bio-nano-chip (BNC), a microfluidic device composed of polydimethylsiloxane (PDMS) and thiolene-based optical epoxy which is both cost-effective and suitable for high performance immunoassays. A novel room temperature (RT) bonding technique was utilized so as to achieve irreversible covalent bonding between PDMS and thiolene-based epoxy layers, while at the same time being compatible with the insertion of agarose bead sensors, selectively arranged in an array of pyramidal microcavities replicated in the thiolene thin film layer. In the sealed device, the bead-supporting epoxy film is sandwiched between two PDMS layers comprising of fluidic injection and drain channels. The agarose bead sensors used in the device are sensitized with anti-C-reactive protein (CRP) antibody, and a fluorescent sandwich-type immunoassay was run to characterize the performance of this device. Computational fluid dynamics (CFD) was used based on the device specifications to model the bead penetration. Experimental data revealed analyte penetration of the immunocomplex to 100 μm into the 280 μm diameter agarose beads, which correlated well with the simulation. A dose-response curve was obtained and the linear dynamic range of the assay was established over 1 ng/mL to 50 ng/mL with a limit of detection less than 1 ng/mL. PMID:21852104

  4. In vivo bioengineered ovarian tumors based on collagen, matrigel, alginate and agarose hydrogels: a comparative study

    International Nuclear Information System (INIS)

    Scaffold-based tumor engineering is rapidly evolving the study of cancer progression. However, the effects of scaffolds and environment on tumor formation have seldom been investigated. In this study, four types of injectable hydrogels, namely, collagen type I, Matrigel, alginate and agarose gels, were loaded with human ovarian cancer SKOV3 cells and then injected into nude mice subcutaneously. The growth of the tumors in vitro was also investigated. After four weeks, the specimens were harvested and analyzed. We found that tumor formation by SKOV3 cells was best supported by collagen, followed by Matrigel, alginate, control (without scaffold) and agarose in vivo. The collagen I group exhibited a larger tumor volume with increased neovascularization and increased necrosis compared with the other materials. Further, increased MMP activity, upregulated expression of laminin and fibronectin and higher levels of HIF-1α and VEGF-A in the collagen group revealed that the engineered tumor is closer to human ovarian carcinoma. In order, collagen, Matrigel, alginate, control (without scaffold) and agarose exhibited decreases in tumor formation. All evidence indicated that the in vivo engineered tumor is scaffold-dependent. Bioactive hydrogels are superior to inert hydrogels at promoting tumor regeneration. In particular, biomimetic hydrogels are advantageous because they provide a microenvironment that mimics the ECM of natural tumors. On the other hand, typical features of cancer cells and the expression of genes related to cancer malignancy were far less similar to the natural tumor in vitro, which indicated the importance of culture environment in vivo. Superior to the in vitro culture, nude mice can be considered satisfactory in vivo ‘bioreactors’ for the screening of favorable cell vehicles for tumor engineering in vitro. (paper)

  5. Acoustic transfer of protein crystals from agarose pedestals to micromeshes for high-throughput screening

    Energy Technology Data Exchange (ETDEWEB)

    Cuttitta, Christina M. [Brookhaven National Laboratory, Upton, NY 11973-5000 (United States); The City University of New York, 2800 Victory Boulevard, Staten Island, NY 10314 (United States); Ericson, Daniel L. [Brookhaven National Laboratory, Upton, NY 11973-5000 (United States); University at Buffalo, SUNY, 12 Capen Hall, Buffalo, NY 14260 (United States); Scalia, Alexander [Brookhaven National Laboratory, Upton, NY 11973-5000 (United States); Binghamton University, 4400 Vestal Parkway East, Binghamton, NY 11973-5000 (United States); Roessler, Christian G. [Brookhaven National Laboratory, Upton, NY 11973-5000 (United States); Teplitsky, Ella [Brookhaven National Laboratory, Upton, NY 11973-5000 (United States); Stony Brook University, Stony Brook, NY 11794-5215 (United States); Joshi, Karan [Brookhaven National Laboratory, Upton, NY 11973-5000 (United States); PEC University of Technology, Chandigarh (India); Campos, Olven [Brookhaven National Laboratory, Upton, NY 11973-5000 (United States); Florida Atlantic University, 777 Glades Road, Boca Raton, FL 33414 (United States); Agarwal, Rakhi; Allaire, Marc [Brookhaven National Laboratory, Upton, NY 11973-5000 (United States); Orville, Allen M. [Brookhaven National Laboratory, Upton, NY 11973-5000 (United States); Brookhaven National Laboratory, Upton, NY 11973-5000 (United States); Sweet, Robert M.; Soares, Alexei S., E-mail: soares@bnl.gov [Brookhaven National Laboratory, Upton, NY 11973-5000 (United States)

    2015-01-01

    An acoustic high-throughput screening method is described for harvesting protein crystals and combining the protein crystals with chemicals such as a fragment library. Acoustic droplet ejection (ADE) is an emerging technology with broad applications in serial crystallography such as growing, improving and manipulating protein crystals. One application of this technology is to gently transfer crystals onto MiTeGen micromeshes with minimal solvent. Once mounted on a micromesh, each crystal can be combined with different chemicals such as crystal-improving additives or a fragment library. Acoustic crystal mounting is fast (2.33 transfers s{sup −1}) and all transfers occur in a sealed environment that is in vapor equilibrium with the mother liquor. Here, a system is presented to retain crystals near the ejection point and away from the inaccessible dead volume at the bottom of the well by placing the crystals on a concave agarose pedestal (CAP) with the same chemical composition as the crystal mother liquor. The bowl-shaped CAP is impenetrable to crystals. Consequently, gravity will gently move the crystals into the optimal location for acoustic ejection. It is demonstrated that an agarose pedestal of this type is compatible with most commercially available crystallization conditions and that protein crystals are readily transferred from the agarose pedestal onto micromeshes with no loss in diffraction quality. It is also shown that crystals can be grown directly on CAPs, which avoids the need to transfer the crystals from the hanging drop to a CAP. This technology has been used to combine thermolysin and lysozyme crystals with an assortment of anomalously scattering heavy atoms. The results point towards a fast nanolitre method for crystal mounting and high-throughput screening.

  6. Subpopulations of liver coated vesicles resolved by preparative agarose gel electrophoresis

    International Nuclear Information System (INIS)

    Rat liver clathrin coated vesicles (CVs) were separated into several distinct subpopulations using non-sieving concentrations of agarose, which allowed the separation of species differing primarily in surface charge. Using preparative agarose electrophoresis, the CVs were recovered and analyzed for differences in morphology, coat protein composition, and stripped vesicle protein composition. Coat proteins from difference populations appeared identical on SDS PAGE, and triskelions stripped from the different populations showed the same mobility on the agarose gel, suggesting that the mobility differences observed in intact CVs were due to differences in the surface charge of underlying vesicles rather than to variations in their clathrin coats. Stripped CVs exhibited considerable heterogeneity when analyzed by Western blotting: the fast-migrating population was enriched in the mannose 6-phosphate receptor, secretory acetyl-choline esterase, and an M/sub r/ 195,000 glycoprotein. The slow-migrating population of CVs was enriched in the asialoglycoprotein receptor, and it appeared to contain all detectable concanavalin A-binding polypeptides as well as the bulk of detectable WGA-binding proteins. When CVs were prepared from 125I-asialoorosomucoid-perfused rat liver, ligand was found in the slow-migrating CVs, suggesting that these were endocytic in origin. Morphological differences were also observed: the fast-migrating population was enriched in smaller CVs, whereas the slow-migrating population exhibited an enrichment in larger CVs. As liver consists largely of hepatocytes, these subpopulations appear to originate from the same cell type and probably represent CVs of different intracellular origin and destination

  7. Studies of transferin polymorphism in Swedish cattle using agarose gel electrophoresis

    International Nuclear Information System (INIS)

    The polymorphic transferrin picture in the sera from 894 Swedish cattle was investigated with an agarose gel electrophoresis technique. The serum transferrin bands in the electrophoresis pattern were first identified by labelling with 59Fe. Six existing phenotypes based on the alleles Tf(supA), Tf(supD) and Tf(supE) could be detected. The frequencies of transferrin types and transferrin alleles are presented, and it is concluded that there are great differences in the frequencis between the Swedish Red and White and the Swedish Friesian. (author)

  8. Sizing of the Haemophilus influenzae Rd genome by pulsed-field agarose gel electrophoresis.

    OpenAIRE

    Lee, J J; Smith, H O

    1988-01-01

    The four restriction enzymes ApaI (5'-GGGCCC), EagI (5'-CGGCCG), NaeI (5'-GCCGGC), and SmaI (5'-CCCGGG) were found to produce distributions of DNA fragment sizes useful for mapping of the Haemophilus influenzae Rd genome by pulsed-field agarose gel electrophoresis. ApaI produced 21 fragments (range, 1.6 to 305 kilobases [kb]), EagI yielded 30 fragments (0.6 to 339 kb), NaeI produced 32 fragments (2.3 to 290 kb), and SmaI yielded 16 fragments (6.0 to 377 kb). Summation of the fragment lengths ...

  9. Micropatterning of a stretchable conductive polymer using inkjet printing and agarose stamping

    DEFF Research Database (Denmark)

    Hansen, Thomas Steen; Hassager, Ole; Larsen, Niels Bent;

    2007-01-01

    A highly conducting stretchable polymer material has been patterned using additive inkjet printing and by subtractive agarose stamping of a deactivation agent (hypochlorite). The material consisted of elastomeric polyurethane combined in an interpenetrating network with a conductive polymer, poly(3....... Inkjet printing of the material was only possible if a short-chain polyurethane was used as elastomer to overcome strain hardening at the neck of the droplets produced for printing. Reproducible line widths down to 200 μm could be achieved by inkjet printing. Both methods were used to fabricate test...

  10. The preparation of low electroendosmosis agarose and its physico-chemical property

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Studies on Gelidium amansii agar fractionations were carried out in this paper. Gelidium amansii agar was fractionated on DEAE-Cellulose, and four fractions were obtained sequentially. The fractions were analyzed on physical and chemical properties, and IR and 13C-NMR spectroscopy applied for elucidating the chemical structure. Among the four fractions obtained, water fraction measured up to the standard of low EEO agarose. The sulfate content, ash content, electroendosmosis and gel strength(1%) of water fraction were 0.16%, 0.34%, 0.12 and 1 130g/cm2 respectively, similar to those of the Sigma products.

  11. Dose response and fading characteristics of an alanine-agarose gel

    International Nuclear Information System (INIS)

    The dose response of an alanine-agarose gel, analyzed by ESR spectrometry, and the stability of the radiation-induced free radicals have been investigated. The stability of the ESR signal is higher for dosimeter samples analyzed at 77 K than for dried samples, analyzed at room-temperature. The dose response is linear to within ±2% in the absorbed dose interval 2-100 Gy. The variations in spectral line shape were analyzed at temperatures between 77 and 270 K. The experimental ESR spectrum at 77 K was compared with a simulated spectrum of polycrystals of L-α-alanine. (Author)

  12. Streptavidin Capture and Detection Using Individual Agarose Bead-based Microfluidic Immunoassay Devices

    Institute of Scientific and Technical Information of China (English)

    Xiaoguang Du

    2009-01-01

    @@ A single microwell on polycarbonate substratc was fabricated using hot embossing by silicon master.The silicon master (85 μm in top,100 μm in bottom,53 μm in height) and 0.25 mm-thick polycarbonate substrate were sandwiched between two glass plates in hot embossing system.The system was heated to 155-160℃ and pressed with a force of 300 psi for 10-30 s.The single microwell was stampted on polycarbonate substrate.Apply a~0.2 μL aliquot of agarose beads to the single microwell.

  13. Effects of surfactants on agarose-based magnetic polymer electrolyte for dye-sensitized solar cells

    International Nuclear Information System (INIS)

    Highlights: ► A novel agarose magnetic polymer electrolyte for DSSC was investigated. ► Four surfactants were introduced to improve the dispersivity of Fe3O4 nanoparticle. ► Fe3O4 nanoparticles are well dispersed and the ionic conductivity was improved. ► TW-80 was selected as the proper surfactant for magnetic polymer electrolyte. -- Abstract: Four surfactants, sodium dodecyl sulfate (SDS), polyvinylpyrrolidone (PVP), polyethylene glycol (PEG200) and polysorbate 80 (TW-80), were added to disperse Fe3O4 nanoparticles in agarose based magnetic polymer electrolyte, for the purpose of improving the performance of dye-sensitized solar cell (DSSC). Fourier transform infrared spectroscopy (FTIR) was employed to characterize the interactions between surfactants and magnetic polymer electrolyte. TW-80 and PEG200 showed good dispersion properties according to surface morphology tests. Through electrochemical impedance spectroscopy (EIS) study, the ionic conductivity, charge transfer resistance, charge recombination resistance and electron lifetime of polymer electrolytes were all improved by modification, while TW-80 modified electrolyte reached the highest ionic conductivity of 2.98 × 10−3 S/cm. Moreover, the photoelectric properties were also significantly enhanced and the best energy conversion efficiency achieved 1.83% with TW-80 modification

  14. Finite difference time domain model of ultrasound propagation in agarose scaffold containing collagen or chondrocytes.

    Science.gov (United States)

    Inkinen, Satu I; Liukkonen, Jukka; Malo, Markus K H; Virén, Tuomas; Jurvelin, Jukka S; Töyräs, Juha

    2016-07-01

    Measurement of ultrasound backscattering is a promising diagnostic technique for arthroscopic evaluation of articular cartilage. However, contribution of collagen and chondrocytes on ultrasound backscattering and speed of sound in cartilage is not fully understood and is experimentally difficult to study. Agarose hydrogels have been used in tissue engineering applications of cartilage. Therefore, the aim of this study was to simulate the propagation of high frequency ultrasound (40 MHz) in agarose scaffolds with varying concentrations of chondrocytes (1 to 32 × 10(6) cells/ml) and collagen (1.56-200 mg/ml) using transversely isotropic two-dimensional finite difference time domain method (FDTD). Backscatter and speed of sound were evaluated from the simulated pulse-echo and through transmission measurements, respectively. Ultrasound backscatter increased with increasing collagen and chondrocyte concentrations. Furthermore, speed of sound increased with increasing collagen concentration. However, this was not observed with increasing chondrocyte concentrations. The present study suggests that the FDTD method may have some applicability in simulations of ultrasound scattering and propagation in constructs containing collagen and chondrocytes. Findings of this study indicate the significant role of collagen and chondrocytes as ultrasound scatterers and can aid in development of modeling approaches for understanding how cartilage architecture affects to the propagation of high frequency ultrasound. PMID:27475127

  15. Pravastatin Improves Glucose Regulation and Biocompatibility of Agarose Encapsulated Porcine Islets following Transplantation into Pancreatectomized Dogs

    Directory of Open Access Journals (Sweden)

    Lawrence S. Gazda

    2014-01-01

    Full Text Available The encapsulation of porcine islets is an attractive methodology for the treatment of Type I diabetes. In the current study, the use of pravastatin as a mild anti-inflammatory agent was investigated in pancreatectomized diabetic canines transplanted with porcine islets encapsulated in agarose-agarose macrobeads and given 80 mg/day of pravastatin (n=3 while control animals did not receive pravastatin (n=3. Control animals reached preimplant insulin requirements on days 18, 19, and 32. Pravastatin-treated animals reached preimplant insulin requirements on days 22, 27, and 50. Two animals from each group received a second macrobead implant: control animals remained insulin-free for 15 and 21 days (AUC = 3003 and 5078 mg/dL/24 hr days 1 to 15 and reached preimplant insulin requirements on days 62 and 131. Pravastatin treated animals remained insulin-free for 21 and 34 days (AUC = 1559 and 1903 mg/dL/24 hr days 1 to 15 and reached preimplant insulin requirements on days 38 and 192. Total incidence (83.3% versus 64.3% and total severity (22.7 versus 18.3 of inflammation on tissue surfaces were higher in the control group at necropsy. These findings support pravastatin therapy in conjunction with the transplantation of encapsulated xenogeneic islets for the treatment of diabetes mellitus.

  16. Dependence of light attenuation and backscattering on collagen concentration and chondrocyte density in agarose scaffolds

    International Nuclear Information System (INIS)

    Optical coherence tomography (OCT) has been applied for high resolution imaging of articular cartilage. However, the contribution of individual structural elements of cartilage on OCT signal has not been thoroughly studied. We hypothesize that both collagen and chondrocytes, essential structural components of cartilage, act as important light scatterers and that variation in their concentrations can be detected by OCT through changes in backscattering and attenuation. To evaluate this hypothesis, we established a controlled model system using agarose scaffolds embedded with variable collagen concentrations and chondrocyte densities. Using OCT, we measured the backscattering coefficient (µb) and total attenuation coefficient (µt) in these scaffolds. Along our hypothesis, light backscattering and attenuation in agarose were dependent on collagen concentration and chondrocyte density. Significant correlations were found between µt and chondrocyte density (ρ = 0.853, p < 0.001) and between µt and collagen concentration (ρ = 0.694, p < 0.001). µb correlated significantly with chondrocyte density (ρ = 0.504, p < 0.001) but not with collagen concentration (ρ = 0.103, p = 0.422) of the scaffold. Thus, quantitation of light backscattering and, especially, attenuation could be valuable when evaluating the integrity of soft tissues, such as articular cartilage with OCT. (paper)

  17. Simulated moving bed separation of agarose-hydrolyzate components for biofuel production from marine biomass.

    Science.gov (United States)

    Kim, Pung-Ho; Nam, Hee-Geun; Park, Chanhun; Wang, Nien-Hwa Linda; Chang, Yong Keun; Mun, Sungyong

    2015-08-01

    The economically-efficient separation of galactose, levulinic acid (LA), and 5-hydroxymethylfurfural (5-HMF) in acid hydrolyzate of agarose has been a key issue in the area of biofuel production from marine biomass. To address this issue, an optimal simulated moving bed (SMB) process for continuous separation of the three agarose-hydrolyzate components with high purities, high yields, and high throughput was developed in this study. As a first step for this task, the adsorption isotherm and mass-transfer parameters of each component on the qualified adsorbent were determined through a series of multiple frontal experiments. The determined parameters were then used in optimizing the SMB process for the considered separation. Finally, the optimized SMB process was tested experimentally using a self-assembled SMB unit with four zones. The SMB experimental results and the relevant computer simulations verified that the developed process in this study was quite successful in the economically-efficient separation of galactose, LA, and 5-HMF in a continuous mode with high purities and high yields. It is thus expected that the developed SMB process in this study will be able to serve as one of the trustworthy ways of improving the economic feasibility of biofuel production from marine biomass. PMID:26141276

  18. Lithium iodide effect on the electrochemical behavior of agarose based polymer electrolyte for dye-sensitized solar cell

    International Nuclear Information System (INIS)

    Highlights: · Conduction behavior in agarose electrolyte system. · Charge recombination resistance is reduced with the increasing LiI concentration. · Charge transfer resistance is also reduced with the increasing LiI concentration. · Electron lifetime is shortened by increasing LiI concentrations. · LiI addition enhances the back reaction in DSSC. - Abstract: The effect of lithium iodide (LiI: 0-85 wt%) on the electrochemical behavior of agarose-based polymer electrolytes for dye-sensitized solar cells (DSSC) was investigated. Fourier Transform Infrared Spectroscopy (FTIR) and scanning electronic microscopy (SEM) were employed to characterize the interactions between polymer matrix and salt and the morphology of the agarose electrolytes, respectively. From the AC impedance spectra study, it was determined that the conduction behavior of the agarose-based polymer electrolyte matches the 'salt-in-polymer' like behavior of low LiI content (0-25 wt%) and 'polymer-in-salt' like behavior of high LiI content (25-85 wt%). Detailed analysis of characteristic electrochemical processes occurring in DSSC with these agarose electrolytes was also obtained by employing the EIS technique. The impedance spectra showed that the electron lifetime of DSSC was shortened with increasing LiI concentration, while the charge transfer resistance and charge recombination resistance were reduced when LiI concentration was increased.

  19. Effect of the hydration on the biomechanical properties in a fibrin-agarose tissue-like model.

    Science.gov (United States)

    Scionti, Giuseppe; Moral, Monica; Toledano, Manuel; Osorio, Raquel; Durán, Juan D G; Alaminos, Miguel; Campos, Antonio; López-López, Modesto T

    2014-08-01

    The effect of hydration on the biomechanical properties of fibrin and fibrin-agarose (FA) tissue-like hydrogels is reported. Native hydrogels with approximately 99.5% of water content and hydrogels with water content reduced until 90% and 80% by means of plastic compression (nanostructuration) were generated. The biomechanical properties of the hydrogels were investigated by tensile, compressive, and shear tests. Experimental results indicate that nanostructuration enhances the biomechanical properties of the hydrogels. This improvement is due to the partial draining of the water that fills the porous network of fibers that the plastic compression generates, which produces a denser material, as confirmed by scanning electron microscopy. Results also indicate that the characteristic compressive and shear parameters increase with agarose concentration, very likely due to the high water holding capacity of agarose, which reduces the compressibility and gives consistency to the hydrogels. However, results of tensile tests indicate a weakening of the hydrogels as agarose concentration increases, which evidences the anisotropic nature of these biomaterials. Interestingly, we found that by adjusting the water and agarose contents it is possible to tune the biomechanical properties of FA hydrogels for a broad range, within which the properties of many native tissues fall. PMID:23963645

  20. Poly-lactic acid and agarose gelatin play an active role in the recovery of spinal cord injury

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    Objective To investigate the role of poly-lactic acid and agarose gelatin in promoting the functional recovery of the injured spinal cord. Methods Poly-lactic acid (PLA) or agarose was embedded in the space between two stumps of the hemisectioned spinal cord. Immunohistochemistry was used to show astroglia proliferation and the infiltration of RhoA-positive cells. Locomotor activity recovery was evaluated by testing the function of hindlimbs. Results Astroglias and RhoA labeled non-neuronal cells accumulated in the area adjacent to the implant, while the number of RhoA-positive cells was decreased dramatically in the absence of implant. Animals implanted with agarose gelatin recovered more quickly than those with PLA, concomitant with a higher survival rate of the neurons. Conclusion Both PLA and agarose gelatin benefited the recovery of spinal cord after injury by providing a scaffold for astroglia processes. Modulation of the rigidity, pore size and inner structure of PLA and agarose gelatin might make these biodegradable materials more effective in the regeneration of the central nervous system (CNS).

  1. Fabrication and Optimization of a PAGATA gel dosimeter: increasing the melting point of the PAGAT gel dosimeter with agarose additive

    International Nuclear Information System (INIS)

    The PAGAT polymer gel dosimeter melts at 30degreeC and even at room temperature during the summer, so it needs to be kept in a cool place such as a refrigerator. To increase the stability of the PAGAT gel, different amounts of agarose were added to the PAGAT gel composition and the PAGATA gel was manufactured. Material and Methods: The PAGATA gel vials were irradiated using a CO-60 machine. Then, the samples were evaluated using a 1.5 T Siemens MRI scanner. The ingredients of the PAGATA normoxic gel dosimeter were 4.5% N-N' methylen-bis-acrylamide, 4.5% acrylamide, 4.5% gelatine, 5 m M tetrakis (THPC), 0.01 mM hydroquinone, 0.5% agarose and 86% de-ionized water (HPLC). Results: Melting point and sensitivity of the PAGAT gel dosimeter with addition of 0.0, 0.3, 0.5, 1.0, 1.5 and 2.0% of agarose were measured, in which the melting points Were increased to 30, 82, 86, 88, 89 and 90degreeC and their sensitivities found to be 0.113, 0.1059, 0.125, 0.122, 0.115 and 0.2 S-1Gy-1 respectively. Discussion and Conclusions: Adding agarose increased the sensitivity and background R2 of the evaluated samples. The optimum amount of agarose was found to be 0.5% regarding these parameters and also the melting point of the gel dosimeter. A value of 0.5% agarose was found to be an optimum value considering the increase of sensitivity to 0.125 and melting point to 86degreeC but at the expense of increasing the background R2 to 4.530.

  2. Comparison of polyacrylamide and agarose gel thin-layer isoelectric focusing for the characterization of beta-lactamases.

    OpenAIRE

    Vecoli, C; Prevost, F E; Ververis, J J; Medeiros, A A; O'Leary, G P

    1983-01-01

    Plasmid-mediated beta-lactamases from strains of Escherichia coli and Pseudomonas aeruginosa were separated by isoelectric focusing on a 0.8-mm thin-layer agarose gel with a pH gradient of 3.5 to 9.5. Their banding patterns and isoelectric points were compared with those obtained with a 2.0-mm polyacrylamide gel as the support medium. The agarose method produced banding patterns and isoelectric points which corresponded to the polyacrylamide gel data for most samples. Differences were observe...

  3. Kinetic model for whey protein hydrolysis by alcalase multipoint-immobilized on agarose gel particles

    Directory of Open Access Journals (Sweden)

    Sousa Jr R.

    2004-01-01

    Full Text Available Partial hydrolysis of whey proteins by enzymes immobilized on an inert support can either change or evidence functional properties of the produced peptides, thereby increasing their applications. The hydrolysis of sweet cheese whey proteins by alcalase, which is multipoint-immobilized on agarose gel, is studied here. A Michaelis-Menten model that takes into account competitive inhibition by the product was fitted to experimental data. The influence of pH on the kinetic parameters in the range 6.0 to 11.0 was assessed, at 50ºC. Initial reaction-rate assays in a pHstat at different concentrations of substrate were used to estimate kinetic and Michaelis-Menten parameters, k and K M. Experimental data from long-term batch assays were used to quantify the inhibition parameter, K I. The fitting of the model to the experimental data was accurate in the entire pH range.

  4. Fractionation of SWNT/nucleic acid complexes by agarose gel electrophoresis

    International Nuclear Information System (INIS)

    We show that aqueous dispersions of single-walled carbon nanotubes (SWNTs), prepared with the aid of nucleic acids (NAs) such as RNA or DNA, can be separated into fractions using agarose gel electrophoresis. In a DC electric field, SWNT/NA complexes migrate in the gel in the direction of positive potential to form well-defined bands. Raman spectroscopy as a function of band position shows that nanotubes having different spectroscopic properties possess different electrophoretic mobilities. The migration patterns for SWNT/RNA and SWNT/DNA complexes differ. Parallel elution of the SWNT/NA complexes from the gel during electrophoresis and subsequent characterization by AFM reveals differences in nanotube diameter, length and curvature. The results suggest that fractionation of nanotubes can be achieved by this procedure. We discuss factors affecting the mobility of the nanotube complexes and propose analytical applications of this technique

  5. Determination of agarose gel pore size: Absorbance measurements vis a vis other techniques

    International Nuclear Information System (INIS)

    The absorbance measurements in the wavelength range 700 nm to 800 nm were used to probe the agarose gel topology evolution and extract the pore size of the trapped solvent. By following the changes in absorbance and pore size, the gelation process could be clearly divided into three stages - induction stage, gelation stage and pseudo-equilibrium stage. The gelation mechanism is explained as a nucleation and growth process. Following the kinetics of gelation using dynamic light scattering is complicated by multiple scattering (for high concentrations) and large fluctuations in intensity and relaxation time. Comparatively, scanning the absorption spectrum is fast and the method is suitable for a wide range of concentrations and setting temperatures. Pore size determination using absorbance is a fast and non-invasive method when compared to the DNA electrophoresis measurements, which extend over several hours and use probe diffusion

  6. Fricke-agarose dosimeter gels: ion diffusion modelling and microdensitometry alternative to MRI

    International Nuclear Information System (INIS)

    Ferric ion diffusion is one of the main problems that still restrains the dosimetric application of Fricke-agarose gels. In this work, we model this process within finite length gel samples. The temporal evolution of the ion concentration as a function of the initial concentration is derived by solving Fick's second law in two dimensions with boundary reflections. The influence of ion concentration gradient, elapsed time, diffusion coefficient and spatial resolution is studied. Due to the main drawbacks of MRI for studying these systems, i.e. high cost and acquisition time often non-negligible compared to diffusion time, we also investigate the possibility of using a microdensitometer. The application of this technique for Fricke gel dosimetry is proposed here for the first time. The estimate of the ion diffusion coefficient is in a very agreement with those reported in literature

  7. Fractionation of SWNT/nucleic acid complexes by agarose gel electrophoresis

    Energy Technology Data Exchange (ETDEWEB)

    Vetcher, Alexandre A [Institute of Biomedical Sciences and Technology and Department of Molecular and Cell Biology, University of Texas at Dallas, Richardson, TX 75083 (United States); Srinivasan, Srimeenakshi [Institute of Biomedical Sciences and Technology and Department of Molecular and Cell Biology, University of Texas at Dallas, Richardson, TX 75083 (United States); Vetcher, Ivan A [Institute of Biomedical Sciences and Technology and Department of Molecular and Cell Biology, University of Texas at Dallas, Richardson, TX 75083 (United States); Abramov, Semen M [NanoTech Institute, University of Texas at Dallas, Richardson, TX 75083 (United States); Kozlov, Mikhail [NanoTech Institute, University of Texas at Dallas, Richardson, TX 75083 (United States); Baughman, Ray H [NanoTech Institute, University of Texas at Dallas, Richardson, TX 75083 (United States); Levene, Stephen D [Institute of Biomedical Sciences and Technology and Department of Molecular and Cell Biology, University of Texas at Dallas, Richardson, TX 75083 (United States)

    2006-08-28

    We show that aqueous dispersions of single-walled carbon nanotubes (SWNTs), prepared with the aid of nucleic acids (NAs) such as RNA or DNA, can be separated into fractions using agarose gel electrophoresis. In a DC electric field, SWNT/NA complexes migrate in the gel in the direction of positive potential to form well-defined bands. Raman spectroscopy as a function of band position shows that nanotubes having different spectroscopic properties possess different electrophoretic mobilities. The migration patterns for SWNT/RNA and SWNT/DNA complexes differ. Parallel elution of the SWNT/NA complexes from the gel during electrophoresis and subsequent characterization by AFM reveals differences in nanotube diameter, length and curvature. The results suggest that fractionation of nanotubes can be achieved by this procedure. We discuss factors affecting the mobility of the nanotube complexes and propose analytical applications of this technique.

  8. Ag@AgI, core@shell structure in agarose matrix as hybrid: synthesis, characterization, and antimicrobial activity.

    Science.gov (United States)

    Ghosh, Somnath; Saraswathi, A; Indi, S S; Hoti, S L; Vasan, H N

    2012-06-01

    A novel in situ core@shell structure consisting of nanoparticles of Ag (Ag Nps) and AgI in agarose matrix (Ag@AgI/agarose) has been synthesized as a hybrid, in order to have an efficient antibacterial agent for repetitive usage with no toxicity. The synthesized core@shell structure is very well characterized by XRD, UV-visible, photoluminescence, and TEM. A detailed antibacterial studies including repetitive cycles are carried out on Gram-negative Escherichia coli (E. coli) and Gram-positive Staphylococcus aureus (S. aureus) bacteria in saline water, both in dark and on exposure to visible light. The hybrid could be recycled for the antibacterial activity and is nontoxic toward human cervical cancer cells (HeLa cells). The water insoluble Ag@AgI in agarose matrix forms a good coating on quartz, having good mechanical strength. EPR and TEM studies are carried out on the Ag@AgI/agarose and the bacteria, respectively, to elucidate a possible mechanism for killing of the bacteria. PMID:22582868

  9. Rapid drug susceptibility test of Mycobacterium tuberculosis using microscopic time-lapse imaging in an agarose matrix.

    Science.gov (United States)

    Choi, Jungil; Yoo, Jungheon; Kim, Ki-Jung; Kim, Eun-Geun; Park, Kyung Ock; Kim, Hyejin; Kim, Haeun; Jung, Hyunju; Kim, Taeyoung; Choi, Myungjin; Kim, Hee Chan; Ryoo, Sungweon; Jung, Yong-Gyun; Kwon, Sunghoon

    2016-03-01

    Tuberculosis (TB) is a major global health problem, and multi-drug-resistant TB (MDR-TB) and extensively drug-resistant TB (XDR-TB) are spreading throughout the world. However, conventional drug susceptibility test (DST) methods, which rely on the detection of the colony formation on a solid medium, require 1-2 months to the result. A rapid and accurate DST is necessary to identify patients with drug-resistant TB and treat them with appropriate drugs. Here, we used microscopic imaging of Mycobacterium tuberculosis (MTB) immobilized in an agarose matrix for a rapid DST. The agarose matrix, which was molded in a microfluidic chip, was inoculated with MTB, and TB drugs in liquid culture medium diffused throughout the agarose to reach the MTB immobilized in the agarose matrix. After the responses of MTB to drugs were tracked with an automated microscopic system, an image-processing program automatically determined the susceptibility and resistance of MTB to specific doses of TB drugs. The automatic DST system was able to assess the drug susceptibility of various drug-resistant clinical TB strains within 9 days with an accuracy comparable to that of conventional method. Our rapid DST method based on microscopic time-lapse imaging greatly reduces the time required for a DST and can be used to rapidly and accurately treat TB patients. PMID:26754815

  10. No Evidence of Viral Transmission following Long-Term Implantation of Agarose Encapsulated Porcine Islets in Diabetic Dogs

    Directory of Open Access Journals (Sweden)

    Lawrence S. Gazda

    2014-01-01

    Full Text Available We have previously described the use of a double coated agarose-agarose porcine islet macrobead for the treatment of type I diabetes mellitus. In the current study, the long-term viral safety of macrobead implantation into pancreatectomized diabetic dogs treated with pravastatin (n=3 was assessed while 2 dogs served as nonimplanted controls. A more gradual return to preimplant insulin requirements occurred after a 2nd implant procedure (days 148, 189, and >652 when compared to a first macrobead implantation (days 9, 21, and 21 in all macrobead implanted animals. In all three implanted dogs, porcine C-peptide was detected in the blood for at least 10 days following the first implant and for at least 26 days following the second implant. C-peptide was also present in the peritoneal fluid of all three implanted dogs at 6 months after 2nd implant and in 2 of 3 dogs at necropsy. Prescreening results of islet macrobeads and culture media prior to transplantation were negative for 13 viruses. No evidence of PERV or other viral transmission was found throughout the study. This study demonstrates that the long-term (2.4 years implantation of agarose-agarose encapsulated porcine islets is a safe procedure in a large animal model of type I diabetes mellitus.

  11. Upregulation of matrix synthesis in chondrocyte-seeded agarose following sustained bi-axial cyclic loading

    Directory of Open Access Journals (Sweden)

    Belinda Pingguan-Murphy

    2012-08-01

    Full Text Available OBJECTIVES: The promotion of extracellular matrix synthesis by chondrocytes is a requisite part of an effective cartilage tissue engineering strategy. The aim of this in vitro study was to determine the effect of bi-axial cyclic mechanical loading on cell proliferation and the synthesis of glycosaminoglycans by chondrocytes in threedimensional cultures. METHOD: A strain comprising 10% direct compression and 1% compressive shear was applied to bovine chondrocytes seeded in an agarose gel during two 12-hour conditioning periods separated by a 12-hour resting period. RESULTS: The bi-axial-loaded chondrocytes demonstrated a significant increase in glycosaminoglycan synthesis compared with samples exposed to uni-axial or no loading over the same period (p<0.05. The use of a free-swelling recovery period prior to the loading regime resulted in additional glycosaminoglycan production and a significant increase in DNA content (p<0.05, indicating cell proliferation. CONCLUSIONS: These results demonstrate that the use of a bi-axial loading regime results in increased matrix production compared with uni-axial loading.

  12. Two-dimensional differential adherence of neuroblasts in laser micromachined CAD/CAM agarose channels

    Energy Technology Data Exchange (ETDEWEB)

    Doraiswamy, A. [Georgia Institute of Technology, School of Material Science and Engineering, Atlanta, GA 30332 (United States); Patz, T. [Georgia Institute of Technology, School of Material Science and Engineering, Atlanta, GA 30332 (United States); Narayan, R.J. [Georgia Institute of Technology, School of Material Science and Engineering, Atlanta, GA 30332 (United States); Dinescu, M. [National Institute for Laser, Plasma and Radiation Physics, P.O. Box MG-16 Magurele, 077125 Bucharest (Romania); Modi, R. [US Naval Research Laboratory, Washington, DC 20375-5345 (United States); Auyeung, R.C.Y. [US Naval Research Laboratory, Washington, DC 20375-5345 (United States); Chrisey, D.B. [US Naval Research Laboratory, Washington, DC 20375-5345 (United States)

    2006-04-30

    Laser micromachining of hydrophobic gels into CAD/CAM patterns was used to develop differentially adherent surfaces and induce the attachment of B35 rat neuroblasts that would later form engineered nerve bundles. Narrow channels, 60-400 {mu}m wide, were micromachined in a 2% agarose gel using an ArF laser, and subsequently filled with an extracellular matrix gel. Upon the addition of 1 ml of a 2 x 104 cells/ml neuroblast suspension, the cells selectively adhered to the ECM-lined channels in a non-confluent manner and we monitored their growth at various time points. The adherent neuroblasts were fluorescently imaged with a propidium iodide live/dead assay, which revealed that the cells were alive within the channels. After 72 h growth, the neuroblasts grew, proliferated, and differentiated into nerve bundles. The fully grown 1 cm long nerve bundle organoids maintained an aspect ratio on the order of 100. The results presented in this paper provide the foundation for laser micromachining technique to develop bioactive substrates for development of three-dimensional tissues. Laser micromachining offers rapid prototyping of substrates, excellent resolution, control of pattern depth and dimensions, and ease of fabrication.

  13. Two-dimensional differential adherence of neuroblasts in laser micromachined CAD/CAM agarose channels

    International Nuclear Information System (INIS)

    Laser micromachining of hydrophobic gels into CAD/CAM patterns was used to develop differentially adherent surfaces and induce the attachment of B35 rat neuroblasts that would later form engineered nerve bundles. Narrow channels, 60-400 μm wide, were micromachined in a 2% agarose gel using an ArF laser, and subsequently filled with an extracellular matrix gel. Upon the addition of 1 ml of a 2 x 104 cells/ml neuroblast suspension, the cells selectively adhered to the ECM-lined channels in a non-confluent manner and we monitored their growth at various time points. The adherent neuroblasts were fluorescently imaged with a propidium iodide live/dead assay, which revealed that the cells were alive within the channels. After 72 h growth, the neuroblasts grew, proliferated, and differentiated into nerve bundles. The fully grown 1 cm long nerve bundle organoids maintained an aspect ratio on the order of 100. The results presented in this paper provide the foundation for laser micromachining technique to develop bioactive substrates for development of three-dimensional tissues. Laser micromachining offers rapid prototyping of substrates, excellent resolution, control of pattern depth and dimensions, and ease of fabrication

  14. Uranium (VI) recovery from aqueous medium using novel floating macroporous alginate-agarose-magnetite cryobeads

    Energy Technology Data Exchange (ETDEWEB)

    Tripathi, Anuj, E-mail: chianuj@gmail.com [Nuclear Agriculture and Biotechnology Division, Bhabha Atomic Research Center, Trombay, Mumbai 400085 (India); Melo, Jose Savio, E-mail: jsmelo@barc.gov.in [Nuclear Agriculture and Biotechnology Division, Bhabha Atomic Research Center, Trombay, Mumbai 400085 (India); D' Souza, Stanislaus Francis, E-mail: sfdsouza@barc.gov.in [Nuclear Agriculture and Biotechnology Division, Bhabha Atomic Research Center, Trombay, Mumbai 400085 (India)

    2013-02-15

    Highlights: ► Designing of floating biopolymeric-magnetite cryobeads using cryotropic-gelation. ► Optimization of preparation process and their physico-chemical characterization. ► First study on the floating cryobeads for uranium recovery application. ► Cost effective synthesis and environment-friendly for environmental applications. -- Abstract: This study presents a novel development of a floating polymeric-magnetite cryobead for the recovery of hexavalent uranium from the aqueous sub-surfaces. The alginate-agarose-magnetite cryobeads were synthesized by the process of cryotropic-gelation at subzero-temperature. The physico-chemical properties of cryobeads showed high surface area and high interconnected porosity (∼90%). Low density of these cryobeads explains their floating property in the aqueous medium. The rheological analysis of cryobeads showed its stability and increased stiffness after uranium adsorption. The presence of magnetite nanoparticles in the porous cryobeads facilitates the recovery of these beads by applying an external magnetic field. Maximum uranium adsorption (97 ± 2%) was observed in the pH range of 4.5–5.5. The thermodynamic parameters suggest passive endothermic adsorption behaviour. HCl was found to be an efficient eluent for the uranium desorption. Five repeated cycles for the desorption of uranium from biosorbent showed 69 ± 3% of uranium recovery. These results suggest stability of these novel floating magnetite-cryobeads under environmetal conditions with potential for the recovery of uranium from contaminated aqueous subsurfaces.

  15. Uranium (VI) recovery from aqueous medium using novel floating macroporous alginate-agarose-magnetite cryobeads.

    Science.gov (United States)

    Tripathi, Anuj; Melo, Jose Savio; D'Souza, Stanislaus Francis

    2013-02-15

    This study presents a novel development of a floating polymeric-magnetite cryobead for the recovery of hexavalent uranium from the aqueous sub-surfaces. The alginate-agarose-magnetite cryobeads were synthesized by the process of cryotropic-gelation at subzero-temperature. The physico-chemical properties of cryobeads showed high surface area and high interconnected porosity (≈ 90%). Low density of these cryobeads explains their floating property in the aqueous medium. The rheological analysis of cryobeads showed its stability and increased stiffness after uranium adsorption. The presence of magnetite nanoparticles in the porous cryobeads facilitates the recovery of these beads by applying an external magnetic field. Maximum uranium adsorption (97 ± 2%) was observed in the pH range of 4.5-5.5. The thermodynamic parameters suggest passive endothermic adsorption behaviour. HCl was found to be an efficient eluent for the uranium desorption. Five repeated cycles for the desorption of uranium from biosorbent showed 69 ± 3% of uranium recovery. These results suggest stability of these novel floating magnetite-cryobeads under environmental conditions with potential for the recovery of uranium from contaminated aqueous subsurfaces. PMID:23280054

  16. Evaluation of agarose gel electrophoresis for characterization of silver nanoparticles in industrial products.

    Science.gov (United States)

    Jimenez, Maria S; Luque-Alled, Jose M; Gomez, Teresa; Castillo, Juan R

    2016-05-01

    Agarose gel electrophoresis (AGE) has been used extensively for characterization of pure nanomaterials or mixtures of pure nanomaterials. We have evaluated the use of AGE for characterization of Ag nanoparticles (NPs) in an industrial product (described as strong antiseptic). Influence of different stabilizing agents (PEG, SDS, and sodium dodecylbenzenesulfonate), buffers (TBE and Tris Glycine), and functionalizing agents (mercaptosuccinic acid (TMA) and proteins) has been investigated for the characterization of AgNPs in the industrial product using different sizes-AgNPs standards. The use of 1% SDS, 0.1% TMA, and Tris Glycine in gel, electrophoresis buffer and loading buffer led to the different sizes-AgNPs standards moved according to their size/charge ratio (obtaining a linear relationship between apparent mobility and mean diameter). After using SDS and TMA, the behavior of the AgNPs in the industrial product (containing a casein matrix) was completely different, being not possible their size characterization. However we demonstrated that AGE with LA-ICP-MS detection is an alternative method to confirm the protein corona formation between the industrial product and two proteins (BSA and transferrin) maintaining NPs-protein binding (what is not possible using SDS-PAGE). PMID:26864499

  17. Isoelectric focusing of human von Willebrand factor in urea-agarose gels

    International Nuclear Information System (INIS)

    An analytical technique has been developed for the isoelectric focusing (IEF) of plasma von Willebrand factor (vWF) in agarose gels containing urea. Under these conditions, vWF freely enters the gel and focuses without artifact. The focused vWF is visualized by staining fixed gels with 125I-labeled affinity-purified heterologous antibody. Utilizing a pH gradient of 5.0-6.5, normal vWF in plasma or purified preparations focuses into at least three bands with apparent isoelectric points (pI) between pH 5.7 and 5.9. A reproducible difference in the IEF pattern of vWF has been established between normal plasmas and those of individuals with variant von Willebrand's disease (vWd) type IIA and type IIB. In type IIA, vWF has a distinctly lower pI than normal. This difference may be related to the presence of smaller vWF multimers in IIA plasma because forms of vWF of corresponding size contained in normal cryoprecipitate supernatant have a similar pI. Type IIB von Willebrand factor has a pI intermediate between normal and IIA. Neuraminidase treatment of plasma samples before IEF results in an increase in pI in normal, type IIA, and type IIB vWF. The data suggest that none of the 16 type IIA and 9 IIB plasmas studied here contain significantly decreased amounts of sialic acid

  18. IPN hydrogel nanocomposites based on agarose and ZnO with antifouling and bactericidal properties.

    Science.gov (United States)

    Wang, Jingjing; Hu, Hongkai; Yang, Zhonglin; Wei, Jun; Li, Juan

    2016-04-01

    Nanocomposite hydrogels with interpenetrating polymer network (IPN) structure based on poly(ethylene glycol) methyl ether methacrylate modified ZnO (ZnO-PEGMA) and 4-azidobenzoic agarose (AG-N3) were prepared by a one-pot strategy under UV irradiation. The hydrogels exhibited a highly macroporous spongelike structure, and the pore size decreased with the increase of the ZnO-PEGMA content. Due to the entanglement and favorable interactions between the two crosslinked networks, the IPN hydrogels exhibited excellent mechanical strength and light transmittance. The maximum compressive and tensile strengths of the IPN hydrogels reached 24.8 and 1.98MPa respectively. The transparent IPN hydrogels transmitted more than 85% of visible light at all wavelengths (400-800nm). The IPN hydrogels exhibited anti-adhesive property towards Gram-negative Escherichia coli (E. coli) and Gram-positive Staphylococcus aureus (S. aureus), and the bactericidal activity increased with the ZnO-PEGMA content. The incorporation of ZnO-PEGMA did not reduce the biocompatibility of the IPN hydrogels and all the IPN nanocomposites showed negligible cytotoxicity. The present study not only provided a facile method for preparing hydrogel nanocomposites with IPN structure but also developed a new hydrogel material which might be an excellent candidate for wound dressings. PMID:26838864

  19. Antifouling coatings based on covalently cross-linked agarose film via thermal azide-alkyne cycloaddition.

    Science.gov (United States)

    Xu, Li Qun; Pranantyo, Dicky; Neoh, Koon-Gee; Kang, En-Tang; Teo, Serena Lay-Ming; Fu, Guo Dong

    2016-05-01

    Coatings based on thin films of agarose-poly(ethylene glycol) (Agr-PEG) cross-linked systems are developed as environmentally-friendly and fouling-resistant marine coatings. The Agr-PEG cross-linked systems were prepared via thermal azide-alkyne cycloaddition (AAC) using azido-functionalized Agr (AgrAz) and activated alkynyl-containing poly(2-propiolamidoethyl methacrylate-co-poly(ethylene glycol)methyl ether methacrylate) P(PEMA-co-PEGMEMA) random copolymers as the precursors. The Agr-PEG cross-linked systems were further deposited onto a SS surface, pre-functionalized with an alkynyl-containing biomimetic anchor, dopamine propiolamide, to form a thin film after thermal treatment. The thin film-coated SS surfaces can effectively reduce the adhesion of marine algae and the settlement of barnacle cyprids. Upon covalent cross-linking, the covalently cross-linked Agr-PEG films coated SS surfaces exhibit good stability in flowing artificial seawater, and enhanced resistance to the settlement of barnacle cyprids, in comparison to that of the surfaces coated with physically cross-linked AgrAz films. PMID:26836479

  20. Stabilization of Candida antarctica Lipase B (CALB Immobilized on Octyl Agarose by Treatment with Polyethyleneimine (PEI

    Directory of Open Access Journals (Sweden)

    Sara Peirce

    2016-06-01

    Full Text Available Lipase B from Candida antarctica (CALB was immobilized on octyl agarose (OC and physically modified with polyethyleneimine (PEI in order to confer a strong ion exchange character to the enzyme and thus enable the immobilization of other enzymes on its surface. The enzyme activity was fully maintained during the coating and the thermal stability was marginally improved. The enzyme release from the support by incubation in the non-ionic detergent Triton X-100 was more difficult after the PEI-coating, suggesting that some intermolecular physical crosslinking had occurred, making this desorption more difficult. Thermal stability was marginally improved, but the stability of the OCCALB-PEI was significantly better than that of OCCALB during inactivation in mixtures of aqueous buffer and organic cosolvents. SDS-PAGE analysis of the inactivated biocatalyst showed the OCCALB released some enzyme to the medium during inactivation, and this was partially prevented by coating with PEI. This effect was obtained without preventing the possibility of reuse of the support by incubation in 2% ionic detergents. That way, this modified CALB not only has a strong anion exchange nature, while maintaining the activity, but it also shows improved stability under diverse reaction conditions without affecting the reversibility of the immobilization.

  1. Dense Pellicular Agarose-Glass Beads for Expanded Bed Application: Flow Hydrodynamics and Solid Phase Classifications

    Institute of Scientific and Technical Information of China (English)

    周鑫; 史清洪; 白姝; 孙彦

    2004-01-01

    Two dense pellicular agarose-glass matrices of different sizes and densities, i.e., AG-S and AG-L, have been characterized for their bed expansion behavior, flow hydrodynamics and particle classifications in an expanded bed system. A 26 mm ID column with side ports was used for sampling the liquid-solid suspension during expanded bed operations. Measurements of the collected solid phase at different column positions yielded the particle size and density distribution data. It was found that the composite matrices showed particle size as well as density classifications along the column axis, i.e., both the size and density of each matrix decreased with increasing the axial bed height. Their axial classifications were expressed by a correlation related to both the particle size and density as a function of the dimensionless axial bed height. The correlation was found to fairly describe the solid phase classifications in the expanded bed system. Moreover, it can also be applied to other two commercial solid matrices designed for expanded bed applications.

  2. Uranium (VI) recovery from aqueous medium using novel floating macroporous alginate-agarose-magnetite cryobeads

    International Nuclear Information System (INIS)

    Highlights: ► Designing of floating biopolymeric-magnetite cryobeads using cryotropic-gelation. ► Optimization of preparation process and their physico-chemical characterization. ► First study on the floating cryobeads for uranium recovery application. ► Cost effective synthesis and environment-friendly for environmental applications. -- Abstract: This study presents a novel development of a floating polymeric-magnetite cryobead for the recovery of hexavalent uranium from the aqueous sub-surfaces. The alginate-agarose-magnetite cryobeads were synthesized by the process of cryotropic-gelation at subzero-temperature. The physico-chemical properties of cryobeads showed high surface area and high interconnected porosity (∼90%). Low density of these cryobeads explains their floating property in the aqueous medium. The rheological analysis of cryobeads showed its stability and increased stiffness after uranium adsorption. The presence of magnetite nanoparticles in the porous cryobeads facilitates the recovery of these beads by applying an external magnetic field. Maximum uranium adsorption (97 ± 2%) was observed in the pH range of 4.5–5.5. The thermodynamic parameters suggest passive endothermic adsorption behaviour. HCl was found to be an efficient eluent for the uranium desorption. Five repeated cycles for the desorption of uranium from biosorbent showed 69 ± 3% of uranium recovery. These results suggest stability of these novel floating magnetite-cryobeads under environmetal conditions with potential for the recovery of uranium from contaminated aqueous subsurfaces

  3. Penetration Deep into Tissues of Reactive Oxygen Species Generated in Floating-Electrode Dielectric Barrier Discharge (FE-DBD): in Vitro Agarose Gel Model Mimicking an Open Wound

    CERN Document Server

    Dobrynin, Danil; Friedman, Gary; Fridman, Alexander

    2013-01-01

    In this manuscript we present an in vitro model based on agarose gel that can be used to simulate a dirty, oily, bloody, and morphologically complex surface of, for example, an open wound. We show this models effectiveness in simulating depth of penetration of reactive species generated in plasma deep into tissue of a rat and confirm the penetration depths with agarose gel model. We envision that in the future such a model could be used to study plasma discharges (and other modalities) and minimize the use of live animals: plasma can be optimized on the agarose gel wound model and then finally verified using an actual wound.

  4. Preparation of a novel pH optical sensor using orange (II) based on agarose membrane as support.

    Science.gov (United States)

    Heydari, Rouhollah; Hosseini, Mohammad; Amraei, Ahmadreza; Mohammadzadeh, Ali

    2016-04-01

    A novel and cost effective optical pH sensor was prepared using covalent immobilization of orange (II) indicator on the agarose membrane as solid support. The fabricated optical sensor was fixed into a sample holder of a spectrophotometer instrument for pH monitoring. Variables affecting sensor performance including pH of dye bonding to agarose membrane and dye concentration were optimized. The sensor responds to the pH changes in the range of 3.0-10.0 with a response time of 2.0 min and appropriate reproducibility (RSD ≤ 0.9%). No significant variation was observed on sensor response after increasing the ionic strength in the range of 0.0-0.5M of sodium chloride. Determination of pH using the proposed optical sensor is quick, simple, inexpensive, selective and sensitive in the pH range of 3.0-10.0. PMID:26838857

  5. Single-Cell-Arrayed Agarose Chip for in Situ Analysis of Cytotoxicity and Genotoxicity of DNA Cross-Linking Agents.

    Science.gov (United States)

    Li, Lili; Wang, Weixing; Ding, Mingyu; Luo, Guoan; Liang, Qionglin

    2016-07-01

    Development of approach or device to allow continuous multiple measurements, such as integrating cytotoxic and genotoxic analysis, is quite appealing for study of the drug's activity and mechanism of action or resistance. In this study, a single-cell-arrayed agarose chip system was developed to combine cell cultivation with subsequent in situ analysis of cytotoxicity and genotoxicity of the chemotherapeutic agent. The modified alkaline comet assay coupled with the Live/Dead assay was used to monitor the interstrand cross-links (ICLs) formation and the cytotoxic effects in different glioma cell lines. In addition, the ICL-induced double strand breaks (DSBs) was measured on the chip to reflect the level of ICLs indirectly. Compared with the traditional methods, the microarray agarose device offers higher throughput, reproducibility, and robustness, exhibiting good potential for high-content drug screening. PMID:27269449

  6. Separation of galactose, 5-hydroxymethylfurfural and levulinic acid in acid hydrolysate of agarose by nanofiltration and electrodialysis.

    Science.gov (United States)

    Kim, Jae Hyung; Na, Jeong-Geol; Yang, Ji-Won; Chang, Yong Keun

    2013-07-01

    A two-stage membrane process for the separation of galactose, 5-hydroxymethylfurfural (5-HMF) and levulinic acid (LA) has been proposed. The first step of nanofiltration (NF) is to remove 5-HMF and LA from galactose solution obtained by the hydrolysis of agarose, the main component of red algal galactan for the reduction of its microbial toxicity. 5-HMF and LA are inhibitory to fermentation but at the same time useful compounds themselves with many applications. The second step of electrodialysis (ED) is to separate 5-HMF and LA in the permeate from NF. More than 91% of 5-HMF and up to 62% of LA could be removed from agarose hydrolysate, while galactose was almost completely retained by NF. Further removal of LA was expected to be possible with no loss of galactose by operating the NF process in a diafiltration mode. 5-HMF and LA could be effectively separated from each other by ED. PMID:23672940

  7. Detection of a single base exchange in PCR-amplified DNA fragments using agarose gel electrophoresis containing bisbenzimide-PEG.

    OpenAIRE

    Müller, M; Kruse, L; Tabrett, A M; Barbara, D.J.

    1997-01-01

    Using PCR fragments of known sequences derived from isolates of two related fungal species, simple submarine electrophoresis in agarose gels containing a bisbenzimide-PEG conjugate (H.A.-Yellow) has been shown to be capable of distinguishing DNA fragments 567 bp long which differ by as little as a single base change. However, only changes affecting bisbenzimide binding sites (which consist of at least four consecutive A/T bases) alter mobility; other changes are ineffective. A second ligand (...

  8. Modification of gel architecture and TBE/TAE buffer composition to minimize heating during agarose gel electrophoresis

    OpenAIRE

    Sanderson, Brian A.; Araki, Naoko; Lilley, Jennifer L.; Guerrero, Gilberto; Lewis, L. Kevin

    2014-01-01

    Agarose gel electrophoresis of DNA and RNA is routinely performed using buffers containing either Tris, acetate and EDTA (TAE) or Tris, borate and EDTA (TBE). Gels are run at a low, constant voltage (~ 10 V/cm) to minimize current and asymmetric heating effects, which can induce band artifacts and poor resolution. In this study, alterations of gel structure and conductive media composition were analyzed to identify factors causing higher electrical currents during horizontal slab gel electrop...

  9. Improved methods for the fluorographic detection of weak β-emitting radioisotopes in agarose and acrylamide gel electrophoresis media

    International Nuclear Information System (INIS)

    The use of acetic acid as a solvent for diphenyloxazole (PPO) in fluorographic procedures has been investigated. It is demonstrated to be superior to both dimethyl sulfoxide and methanol with respect to its suitability in both agarose and acrylamide gel electrophoresis systems. In addition, a method has been developed for impregnating fragile gels such as those used for immunodiffusion with PPO in preparation for fluorography. (Auth.)

  10. DNA fragmentation of human infarcted myocardial cells demonstrated by the nick end labeling method and DNA agarose gel electrophoresis.

    OpenAIRE

    Itoh, G; Tamura, J; M. Suzuki; Suzuki, Y.; Ikeda, H; Koike, M; Nomura, M; Jie, T; Ito, K

    1995-01-01

    Myocardial tissue taken from 19 autopsy cases of myocardial infarction were examined both by the nick and labeling method (NELM) and by DNA agarose gel electrophoresis in order to demonstrate the localization of cells with fragmented DNA and to confirm the internucleosomal cleavage of DNA biochemically. The nuclei corresponding to those with the histological features of acute myocardial infarction in hematoxylin and eosin (H&E)-stained sections were stained strongly positive with the nick end...

  11. Pulse time and agarose concentration affect the electrophoretic mobility of cccDNA during PFGE and FIGE [corrected].

    OpenAIRE

    Sobral, B W; Atherly, A G

    1989-01-01

    Circular DNAs have been shown to migrate in an unusual manner during field inversion gel electrophoresis (FIGE) and orthogonal field alternating gel electrophoresis (OFAGE). We studied the effect of varying pulse time and agarose concentration on the electrophoretic mobility of supercoiled (ccc) DNAs ranging from 2 kbp to 16 kbp during FIGE and contoured homogeneous electric fields (CHEF). Both supercoiled and linear molecules display a minimum mobility as a function of pulse time in a CHEF a...

  12. Continuous Production of 6-amino Penicillanic Acid (6-APA) by Agarose Immobilized Penicillin Acylase in a Packed Column Reactor

    OpenAIRE

    Banerjee, S.; Debnath, M.

    2007-01-01

    Penicillin acylase, an industrially important biocatalyst catalyzes the conversion of penicillins to 6-amino penicillanic acid (6-APA) which is the main precursor for the production of semi-synthetic -lactam antibiotics. The present work involves the continuous production of 6-APA in a packed column reactor by using agarose immobilized penicillin acylase as a block polymer. The strain Escherichia coli ATCC 11105 was used as enzyme source and penicillin G as substrate. The acidic nature of 6-A...

  13. Combination of fibrin-agarose hydrogels and adipose-derived mesenchymal stem cells for peripheral nerve regeneration

    Science.gov (United States)

    Carriel, Víctor; Garrido-Gómez, Juan; Hernández-Cortés, Pedro; Garzón, Ingrid; García-García, Salomé; Sáez-Moreno, José Antonio; Sánchez-Quevedo, María del Carmen; Campos, Antonio; Alaminos, Miguel

    2013-04-01

    Objective. The objective was to study the effectiveness of a commercially available collagen conduit filled with fibrin-agarose hydrogels alone or with fibrin-agarose hydrogels containing autologous adipose-derived mesenchymal stem cells (ADMSCs) in a rat sciatic nerve injury model. Approach. A 10 mm gap was created in the sciatic nerve of 48 rats and repaired using saline-filled collagen conduits or collagen conduits filled with fibrin-agarose hydrogels alone (acellular conduits) or with hydrogels containing ADMSCs (ADMSC conduits). Nerve regeneration was assessed in clinical, electrophysiological and histological studies. Main results. Clinical and electrophysiological outcomes were more favorable with ADMSC conduits than with the acellular or saline conduits, evidencing a significant recovery of sensory and motor functions. Histological analysis showed that ADMSC conduits produce more effective nerve regeneration by Schwann cells, with higher remyelination and properly oriented axonal growth that reached the distal areas of the grafted conduits, and with intensely positive expressions of S100, neurofilament and laminin. Extracellular matrix was also more abundant and better organized around regenerated nerve tissues with ADMSC conduits than those with acellular or saline conduits. Significance. Clinical, electrophysiological and histological improvements obtained with tissue-engineered ADMSC conduits may contribute to enhancing axonal regeneration by Schwann cells.

  14. In vitro study of using calcium phosphate cement as immunoisolative device to enclose insulinoma/agarose microspheres as bioartificial pancreas.

    Science.gov (United States)

    Kai-Chiang, Yang; Ching-Yao, Yang; Chang-Chin, Wu; Tzong-Fu, Kuo; Feng-Huei, Lin

    2007-12-15

    In this study, the feasibility of using calcium phosphate cement (CPC) as immunoisolative device to enclose insulinoma/agarose microspheres as bioartificial pancreas was evaluated. We fabricated a chamber by CPC and utilized X-ray diffraction, Scanning electron microscope and Mercury intrusion porosimetry to identify the characters of the CPC chamber. The nominal molecular weight cut-off and cytotoxicity of CPC chamber were also evaluated. An insulinoma cell line (RIN-m5F) was chosen as insulin source and encapsulated in agarose microspheres and then enclosed in preformed CPC chamber. Insulin secretion was analyzed by Enzyme-linked immunosorbant assay to evaluate the function of insulinoma enclosed in CPC chamber. Results showed that the CPC chamber was non-cytotoxicity to insulinoma and can block the penetration of molecules which molecular weight larger than 12.4 kDa. Insulinoma inside the CPC chamber can secrete insulin in stable level for 30 days. This study indicated that we may use CPC as immunoisolative material to enclose insulinoma/agarose microspheres as bioartificial pancreas. PMID:17514757

  15. [Agarose gel isoelectric focusing: application to the study of abnormalities of immunoglobulin clonality in CSF and serum].

    Science.gov (United States)

    Lebrun-Fourcy, C; Rondot, J; Revol, C; Renversez, J C

    1996-01-01

    Since it is quite difficult to commonly use isoelectric focusing (IEF) of proteins in polyacrylamide gel for biological diagnosis, we have developed a method based on IEF in agarose gel, to split proteins from sera and cerebrospinal fluid (CSF). A prefocalisation at low voltage (250 V) is made on a custom thin gel of agarose (0.5 mm) containing some carrier ampholytes (pH 5-9). After deposition of biological samples, the gel is run at 500 V, thereafter at 1200 V. After focusing, the gel is fixed before being coloured by a simplified silver staining technique. In order to demonstrate the good resolution of the immunoglobulines (Ig) in the pH gradient, a transfer on a nitrocellulose membrane followed by an immunofixation was carried out from unstained gels after IEF. This separation on agarose gel shows several advantages, ie its speed (3H total), its lack of toxicity, its sensibility and its reproductibility. It is specially well suited for the diagnosis of diseases characterised by oligoclonal or monoclonal Ig, particularly those found in the CSF during neurologic diseases like multiple sclerosis. Several examples of focused sera and CSF are reviewed in the paper. PMID:8952725

  16. Effect of saccharide additives on response of ferrous-agarose-xylenol orange radiotherapy gel dosimeters

    International Nuclear Information System (INIS)

    Glucose, sucrose, starch, and locust bean gum have been used as additives to the ferrous-agarose-xylenol orange (FAX) gel dosimeter. The saccharide enhanced dosimeters were found to have a higher dose sensitivity over a standard FAX gel as measured by both optical density change and magnetic resonance imaging (MRI). With optical density measurement, OD-dose sensitivity increases were up to 55% for glucose, 122% for sucrose and 43% for starch, while locust bean gum did not give a consistent response. With MRI, R1-dose sensitivity increases were up to 178% with sucrose addition. The FAX gel with sucrose was studied in greatest detail. The OD-dose sensitivity dependence on cooling rate was reduced for the sucrose FAX gel over the standard FAX gel, which has significant implications for uniform dose sensitivity in large gel phantoms. The thermal oxidation rate in the sucrose FAX gel was up to 2.3 times higher than in the standard gel. The OD-dose sensitivity of oxygenated sucrose FAX gels was 4.3 times greater than standard FAX gels, while continued enhancement in OD-dose sensitivity with increased sucrose concentrations beyond 2.0 g/l was found only for the oxygenated sucrose FAX gels. Both the molar absorption coefficient of the ferric ion-xylenol orange complex at 543 nm and gel pH were not affected by the presence of sucrose, with the implication that the higher OD-dose sensitivity of gels with saccharides is due to increased chain reaction production of ferric ions

  17. Recording of radiation-induced optical density changes in doped agarose gels with a CCD camera

    International Nuclear Information System (INIS)

    Full text: Spatially resolved dose measurement with iron-doped agarose gels is continuing to be investigated for applications in radiotherapy dosimetry. It has previously been proposed to use optical methods, rather than MRI, for dose measurement with such gels and this has been investigated using a spectrophotometer (Appleby A and Leghrouz A, Med Phys, 18:309-312, 1991). We have previously studied the use of a pencil beam laser for such optical density measurement of gels and are currently investigating charge-coupled devices (CCD) camera imaging for the same purpose but with the advantages of higher data acquisition rates and potentially greater spatial resolution. The gels used in these studies were poured, irradiated and optically analysed in Perspex casts providing gel sections 1 cm thick and up to 20 cm x 30 cm in dimension. The gels were also infused with a metal indicator dye (xylenol orange) to render the radiation induced oxidation of the iron in the gel sensitive to optical radiation, specifically in the green spectral region. Data acquisition with the CCD camera involved illumination of the irradiated gel section with a diffuse white light source, with the light from the plane of the gel section focussed to the CCD array with a manual zoom lens. The light was also filtered with a green colour glass filter to maximise the contrast between unirradiated and irradiated gels. The CCD camera (EG and G Reticon MC4013) featured a 1024 x 1024 pixel array and was interfaced to a PC via a frame grabber acquisition board with 8 bit resolution. The performance of the gel dosimeter was appraised in mapping of physical and dynamic wedged 6 MV X-ray fields. The results from the CCD camera detection system were compared with both ionisation chamber data and laser based optical density measurements of the gels. Cross beam profiles were extracted from each measurement system at a particular depth (eg. 2.3 cm for the physical wedge field) for direct comparison. A

  18. Comparative analysis of thyroid extract gel filtration by dextran gel (Sephadex G-200) and agarose (Sepharose 6-B)

    International Nuclear Information System (INIS)

    Separation of thyroglobulin and havier proteins from crude thyroid gland extracts using molecular through agarose gel (Sepharose-6B) is done. In order to compare the separation obtained on Sephadex wiht that on Sepharose, parallel filtrations are run with extratcts from two thyroid adenomas, one 'cold' and one 'hot' nodule, and their normal contralateral tissues. On Sephadex, good separation is ibtained between the heavy proteins and thyroglobulin, separation between thyroglobulin and proteins is better ou Sephacex than on Sepharose althrough, due to the smaller diluition which the lighter fraction suffers on Sephadex, an efficient qualitative analysis is possible

  19. Topological complexity of different populations of pBR322 as visualized by two-dimensional agarose gel electrophoresis

    OpenAIRE

    Martin-Parras, Luis; Lucas, Isabelle A.; Martínez-Robles, María Luisa; Hernandez, Pablo; Krimer, Dora B.; Hyrien, Olivier; Schvartzman, Jorge Bernardo

    1998-01-01

    Neutral/neutral two-dimensional (2D) agarose gel electrophoresis was used to investigate populations of the different topological conformations that pBR322 can adopt in vivo in bacterial cells as well as in Xenopus egg extracts. To help in interpretation and identification of all the different signals, undigested as well as DNA samples pretreated with DNase I, topoisomerase I and topoisomerase II were analyzed. The second dimension of the 2D gel system was run with or without ethidium bromide...

  20. The migration behaviour of DNA replicative intermediates containing an internal bubble analyzed by two-dimensional agarose gel electrophoresis

    OpenAIRE

    Schvartzman, Jorge Bernardo; Martínez-Robles, María Luisa; Hernandez, Pablo

    1993-01-01

    Initiation of DNA replication in higher eukaryotes is still a matter of controversy. Some evidence suggests it occurs at specific sites. Data obtained using two-dimensional (2D) agarose gel electrophoresis, however, led to the notion that it may occur at random in broad zones. This hypothesis is primarily based on the observation that several contiguous DNA fragments generate a mixture of the so-called 'bubble' and 'simple Y' patterns in Neutral/neutral 2D gels. The interpretation that this m...

  1. Silver nanoparticles doped agarose disk: highly sensitive surface-enhanced Raman scattering substrate for in situ analysis of ink dyes.

    Science.gov (United States)

    Raza, Ali; Saha, Basudeb

    2013-12-10

    Raman spectroscopy is a preferred analytical tool for forensic trace analysis due to its non-invasive nature. This technique has been utilized in examination of organic colorants present in fibers and ink, but high fluorescent nature of these compounds is a problem. In the present study, silver-doped agarose gel disk, having property of quenching fluorescence and enhancing Raman signals, is found to be effective as surface-enhanced Raman scattering (SERS) substrates for analysis of rhodamine 6G (Rh 6G) and crystal violet (CV) dyes. As-prepared and well characterized by UV, TEM-EDAX and XRD techniques, the investigated silver-doped agarose gel disk proves to have minimal invasive as confirmed by the ATR-FTIR method and effective for in situ SERS analysis of blue and red ballpoint ink. The disk is stable upon storage and hence can be re-used and re-examined. The present method offers new possibilities in trace forensic analysis with minimal destruction. PMID:24314497

  2. Determining iron oxide nanoparticle heating efficiency and elucidating local nanoparticle temperature for application in agarose gel-based tumor model.

    Science.gov (United States)

    Shah, Rhythm R; Dombrowsky, Alexander R; Paulson, Abigail L; Johnson, Margaret P; Nikles, David E; Brazel, Christopher S

    2016-11-01

    Magnetic iron oxide nanoparticles (MNPs) have been developed for magnetic fluid hyperthermia (MFH) cancer therapy, where cancer cells are treated through the heat generated by application of a high frequency magnetic field. This heat has also been proposed as a mechanism to trigger release of chemotherapy agents. In each of these cases, MNPs with optimal heating performance can be used to maximize therapeutic effect while minimizing the required dosage of MNPs. In this study, the heating efficiencies (or specific absorption rate, SAR) of two types of MNPs were evaluated experimentally and then predicted from their magnetic properties. MNPs were also incorporated in the core of poly(ethylene glycol-b-caprolactone) micelles, co-localized with rhodamine B fluorescent dye attached to polycaprolactone to monitor local, nanoscale temperatures during magnetic heating. Despite a relatively high SAR produced by these MNPs, no significant temperature rise beyond that observed in the bulk solution was measured by fluorescence in the core of the magnetic micelles. MNPs were also incorporated into a macro-scale agarose gel system that mimicked a tumor targeted by MNPs and surrounded by healthy tissues. The agarose-based tumor models showed that targeted MNPs can reach hyperthermia temperatures inside a tumor with a sufficient MNP concentration, while causing minimal temperature rise in the healthy tissue surrounding the tumor. PMID:27523991

  3. Probing the transport of plasma-generated RONS in an agarose target as surrogate for real tissue: dependency on time, distance and material composition

    International Nuclear Information System (INIS)

    We report a simple experimental approach to follow the transport of helium (He) plasma-generated reactive oxygen and nitrogen species (RONS) through millimetre thick agarose targets. These RONS may be either primary RONS, generated directly by the plasma jet, or secondary RONS generated for example at the surface of, or within, the material. Our experiment involves placing an agarose film over a quartz cuvette filled with deionized water. The agarose film is exposed to a He plasma jet and the UV absorption profile (of the deionized water) is recorded in real-time. Plasma exposure time, source-target distance and agarose film thickness and composition are varied to explore their effects on the depth of RONS delivery by the plasma jet. We conclude that plasma UV plays a minor role in the transport of RONS; whereas direct plasma contact and the He gas flow promote the transport of RONS into tissue. Our data indicate an accumulation of RONS within the agarose film (during plasma exposure) and a subsequent (time-lagged) release into the deionized water. Our approach can be readily adapted to other plasma sources; it can accommodate more complex biological materials, and has the potential to provide new insights into plasma-induced phenomena within real tissues. (fast track communication)

  4. Penetration Deep into Tissues of Reactive Oxygen Species Generated in Floating-Electrode Dielectric Barrier Discharge (FE-DBD): in Vitro Agarose Gel Model Mimicking an Open Wound

    OpenAIRE

    Dobrynin, Danil; Fridman, Gregory; Friedman, Gary; Fridman, Alexander

    2013-01-01

    In this manuscript we present an in vitro model based on agarose gel that can be used to simulate a dirty, oily, bloody, and morphologically complex surface of, for example, an open wound. We show this models effectiveness in simulating depth of penetration of reactive species generated in plasma deep into tissue of a rat and confirm the penetration depths with agarose gel model. We envision that in the future such a model could be used to study plasma discharges (and other modalities) and mi...

  5. Effect of encephlitogenic protein. PPD and tetanus toxoid on leukocyte migration in agarose. A study of "cross-reactivity".

    Science.gov (United States)

    Källén, B; Nilsson, O

    1979-04-01

    The reactivity to three antigens: bovine encephalitogenic protein (EP), PPD, and tetanus toxoid, was studied with blood leukocytes from healthy humans using Clausen's (5) leukocyte migration in agarose technique. There is an obvious correlation between the reaction of EP (all concentrations studied) and to low concentrations of PPD; and between the reactivity to low concentrations of EP and low concentrations of tetanus toxoid. After vaccination with tetanus vaccine, a marked increase in reactivity to the toxoid sometimes occurred; at the same time, a marked reactivity to EP appeared. Various explanations are discussed: a true immunological cross-reactivity, that the correlations are due to a variability in individual response with respect to lymphokine production, and that BCG and tetanus vaccinations produce an adjuvant effect increasing a pre-existing low reactivity to EP. PMID:89821

  6. Detection of Lesch-Nyhan syndrome carriers: Analysis of hair roots for HPRT by agarose gel electrophoresis and autoradiography

    International Nuclear Information System (INIS)

    Flat agarose gel electrophoresis and autoradiography were used to analyze hypoxanthine phosphoribosyltransferase (HPRT) and adenine phosphoribosyltransferase (APRT) activity in individual hair roots collected from the scalps of females to determine the presence of HPRT-deficient cells. Autoradiographs of hair-root lysates of normal homozygous females contained two well-separated dark zones representing HPRT and APRT activities. In contrast, some hair roots from carriers of HPRT deficiency contained two zones of activity with the same relative proportion of APRT and HPRT as hair roots of normal homozygotes, while others contained decreased amounts of HPRT activity. These hair roots consisted of HPRT+ and HPRT- cells. In addition, some hair roots from heterozygous carriers contained APRT but no HPRT activity. Such hair roots consisted of HPRT- cells only. (author)

  7. Electron microscopic and agarose gel electrophoretic studies on apoptosis in immune cells induced by enriched 235U

    International Nuclear Information System (INIS)

    At present, the apoptosis in Molt-4 cell (a human acute lymphoblastic leukemia cell line) and Ana-1 cell (a macrophage cell line) were studied after internal irradiation with enriched 235U. The cumulative radiation absorption dose of 235U in cultural cells through different periods were estimated. The morphological changes, which observed by electron microscopy, indicated that Molt-4 and Ana-1 immune cells after incubation with 235U, displayed nuclear fragmentation, margination of condensed chromatin, as well as the membrane-bounded apoptotic bodies formation. The agarose gel electrophoretic observations showed the DNA ladder pattern formation in Molt-4 cell as well as in Ana-1 cell. The experimental results showed that apoptosis induced by 235U in immune cells, were dependent on the 235U-treated time and cumulative radiation absorption dose

  8. Development of a new method for the detection of vanadium complexes bound to DNA, using Agarose Gel Electrophoresis

    OpenAIRE

    Subedi, Prabal

    2012-01-01

    Existem apenas alguns métodos disponíveis para o estudo da ligação de metais ao ADN. Estes são baseados em técnicas espectroscópicas, que podem apenas ser utilizadas quando determinados cromóforos quer da molécula de ADN ou dos complexos metálicos estão directamente envolvidos na ligação de metais ao ADN. O objectivo deste projecto foi desenvolver um novo método que pode ser utilizado para detectar a ligação de um metal de transição ao ADN, utilizando Electroforese em gel de agarose (EGA)...

  9. Synthesis and characterization of macroporous alginate-agarose-magnetite cryobeads for their application in uranium sorption from aqueous medium

    International Nuclear Information System (INIS)

    Contamination of water by heavy metals and radionuclides has become an increasing problem to the environment, which affects the agricultural lands, environmental flora and fauna and importantly human health. There is an interest to develop a simple cost effective technology for the separation of heavy metals from aqueous sub-surfaces. We have developed a novel floating polymeric-magnetite cryobead for the sorption of hexavalent uranium from the aqueous medium. The covalently crosslinked alginate-agarose-magnetite (AAM) cryobeads were synthesized by the process of cryogelation at subzero temperature (i.e. -20 ℃). Alginate polymer was selected for the synthesis of cryobead due to the presence of natural ligand (carboxyl), which interacts with uranyl ions. Agarose was used to provide strength and stability to the cryobeads. Using the AAM cryobeads, we have observed upto 97 % uranium adsorption within 30 min at an initial concentration of 100 mg/L uranium. Due to the macroporous architecture of the cryobeads, the adsorption kinetics was increased 3 folds unlike what has been reported in earlier studies. The study on the effect of pH suggests maximum uranium adsorption (qmax) in the range of 4.5 to 5.5. The thermodynamic parameters i.e. variation in entropy (ΔS), enthalpy (ΔH) and Gibbs free energy (ΔG) were calculated which suggest passive endothermic adsorption behaviour up to 50℃. HCl was found to be an efficient eluent for the uranium desorption. Five repeated cycles for desorption of uranium from biosorbent showed 70 % of uranium recovery. These results suggest stability of novel floating magnetite-cryobeads under acidic conditions and reusability with potential for the recovery of uranium from contaminated aqueous subsurfaces

  10. Evaluation of the friction coefficient, the radial stress, and the damage work during needle insertions into agarose gels.

    Science.gov (United States)

    Urrea, Fabián A; Casanova, Fernando; Orozco, Gustavo A; García, José J

    2016-03-01

    Agarose hydrogels have been extensively used as a phantom material to mimic the mechanical behavior of soft biological tissues, e.g. in studies aimed to analyze needle insertions into the organs producing tissue damage. To better predict the radial stress and damage during needle insertions, this study was aimed to determine the friction coefficient between the material of commercial catheters and hydrogels. The friction coefficient, the tissue damage and the radial stress were evaluated at 0.2, 1.8, and 10mm/s velocities for 28, 30, and 32 gauge needles of outer diameters equal to 0.36, 0.31, and 0.23mm, respectively. Force measurements during needle insertions and retractions on agarose gel samples were used to analyze damage and radial stress. The static friction coefficient (0.295±0.056) was significantly higher than the dynamic (0.255±0.086). The static and dynamic friction coefficients were significantly smaller for the 0.2mm/s velocity compared to those for the other two velocities, and there was no significant difference between the friction coefficients for 1.8 and 10mm/s. Radial stress averages were 131.2±54.1, 248.3±64.2, and 804.9±164.3Pa for the insertion velocity of 0.2, 1.8, and 10mm/s, respectively. The radial stress presented a tendency to increase at higher insertion velocities and needle size, which is consistent with other studies. However, the damage work did not show to be a good predictor of tissue damage, which appears to be due to simplifications in the analytical model. Differently to other approaches, the method proposed here based on radial stress may be extended in future studies to quantity tissue damage in vivo along the entire needle track. PMID:26700572

  11. Separation of human IgG fragments using copper, nickel, zinc, and cobalt chelated to CM-Asp-agarose by positive and negative chromatography.

    Science.gov (United States)

    Mourão, Cecília Alves; Carmignotto, Gabriela Pannunzio; Bueno, Sonia Maria Alves

    2016-04-01

    This study evaluated the feasibility of using immobilized metal-ion affinity chromatography (IMAC) for separation of human Fab fragments using four different transition metal ions copper, nickel, zinc, and cobalt chelated to CM-Asp (carboxymethylaspartate) immobilized on the agarose gel. The Fab and Fc fragments (from human IgG digested with papain) interacted differently with the chelates studied, depending on the adsorption buffer system. The interaction between chelate and Fc fragment is predominantly based on the coordination bonds using adsorption buffer containing NaCl. Negative chromatography was performed on Cu(II)-CM-Asp-agarose obtaining 2.9mg of Fab per mL of adsorbent in nonretained fractions (Fc fragment-free without uncleaved IgG). The adsorption of Fab fragments is governed by electrostatic forces in the absence of NaCl in the adsorption buffer. High selectivity was achieved on Co(II)-CM-Asp-agarose and 5.7mg of Fab per mL of adsorbent was obtained in eluted fractions without Fc fragments, although having uncleaved IgG. The results showed that chromatography on transition metal ions chetated to CM-Asp-agarose is a promising approach to separation of Fab fragments from papain-digested human IgG solution. PMID:26974869

  12. Quantification of DNA by Agarose Gel Electrophoresis and Analysis of the Topoisomers of Plasmid and M13 DNA Following Treatment with a Restriction Endonuclease or DNA Topoisomerase I

    Science.gov (United States)

    Tweedie, John W.; Stowell, Kathryn M.

    2005-01-01

    A two-session laboratory exercise for advanced undergraduate students in biochemistry and molecular biology is described. The first session introduces students to DNA quantification by ultraviolet absorbance and agarose gel electrophoresis followed by ethidium bromide staining. The second session involves treatment of various topological forms of…

  13. The use of nitrocellulose blotting for the study of hepatitis B surface antigen electrophoresed in agarose gels

    International Nuclear Information System (INIS)

    Nitrocellulose-protein blotting of serum electrophoresed in agarose gels has been adapted for the study of hepatitis B surface antigen (HBsAg). 125I-labeled anti-HBs was used as the antigen probe, and the electrophoretic migration was monitored by autoradiography. The method required 3 μl or less of serum and could detect as little as 1 pg of purified HBsAg. Typically, the authors observed two bands of HBsAg; a moving band which migrated about one-third the distance moved by human serum albumin and a non-migratory band which remained at the loading site. Some examples of the use of the method include: (1) empirical methods for correlating HBsAg concentration in serum to film darkness; (2) observations of mobility changes in serial sera from dialysis patients with chronic HBsAg antigenemia; and (3) detection of related antigens such as antigen from the PLC/PRF/5 hepatoma tissue culture line and the cross-reacting woodchuck hepatitis virus surface antigen (WHsAg). (Auth)

  14. Agarose cell block technique as a complementary method in the diagnosis of fungal osteomyelitis in a dog

    Directory of Open Access Journals (Sweden)

    N.S. Rocha

    2012-04-01

    Full Text Available A 7-year-old Labrador Retriever female dog presenting left forelimb lameness for one day was admitted to the Veterinary Hospital (UNESP-Botucatu for clinical evaluation. Several tests, including blood and image analysis, microbiological culture and cytology of lytic areas of affected bone were made in order to establish a diagnosis. Serum biochemical profile revealed increased levels of liver enzymes, plasma globulin, creatine kinase (CK and calcium. Hemogram revealed anemia and leukocytosis; left humerus image analysis revealed an osteolytic lesion and cytology revealed a suppurative periostitis. Differential diagnosis was a nonspecific infectious inflammatory process or osteosarcoma. Since it was not possible to achieve a definitive diagnosis and there was a highly suspicious for an infectious agent, an agarose cell block of the bone marrow fine-needle aspiration was made. The cytological examination of cell block presented similar findings as described previously. However, additional stains including periodic acid-Schiff (PAS were positive for fungal hyphae, which rendered a diagnosis of fungal osteomyelitis due to Aspergillus spp. This case report illustrates an uncommon cause of osteomyelitis for breed that was diagnosed by an underused method in veterinary medicine.

  15. Characterization of β -Glucosidase Produced by Aspergillus niger under Solid-State Fermentation and Partially Purified Using MANAE-Agarose.

    Science.gov (United States)

    Baraldo Junior, Anderson; Borges, Diogo G; Tardioli, Paulo W; Farinas, Cristiane S

    2014-01-01

    β -Glucosidase (BGL) is a hydrolytic enzyme with specificity for a wide variety of glycoside substrates, being an enzyme with a large range of biotechnological applications. However, enzyme properties can be different depending both on the microorganism and the cultivation procedure employed. Therefore, in order to explore potential biocatalytical applications of novel enzymes, their characterization is essential. In this work, a BGL synthesized by a selected strain of Aspergillus niger cultivated under solid-state fermentation (SSF) was partially purified and fully characterized in terms of optimum pH, temperature, and thermostability. The single-step purification using MANAE-agarose in a chromatographic column yielded an enzyme solution with specific activity (17.1 IU/mg protein) adequate for the characterization procedures. Electrophoresis SDS-PAGE and size-exclusion chromatography analysis resulted in an estimated molecular mass of 60 kDa. Higher enzyme activities were found in the range between 40 and 65°C and between pH 4 and 5.5, indicating an interesting characteristic for application in the hydrolysis of lignocellulosic biomass for biofuels production. Thermostability studies of purified BGL resulted in half-lives at 37°C of 56.3 h and at 50°C of 5.4 h. These results provide support for further studies of this enzyme towards revealing its potential biotechnological applications. PMID:24940510

  16. A thin-layer multistrip agarose gel electrophoresis apparatus for Ferguson plot analysis at the sub-microgram load level.

    Science.gov (United States)

    Orbàn, L; Sullivan, J V; Chrambach, A

    1989-07-01

    A method for the simultaneous horizontal agarose gel electrophoresis on thin-layer strips of different gel concentrations was developed for the purpose of generating Ferguson plots at the sub-microgram load level. Seven independent gel strips on a common GelBond support were formed by filling channels created by a comb-shaped spacer (polycarbonate) in a vertical multistrip cassette. Electrophoresis on a horizontal Peltier-cooled surface employed commercial apparatus (E-C Apparatus Corp.) with a modified cover which is airtight and holds anodic and cathodic voltage measurement probes for each strip. The application of the apparatus to Ferguson plot analysis in a single experiment was exemplified on the RNA-containing turnip crinkle virus (TCV) at a load of 50 ng/gel strip, using an optimized silver staining method (a modification of a procedure of FMC Corp. BioProducts) for detection. Within the range of 3.5 to 12.5 V/cm, the plot was found to be independent of field strength. Mobility is also independent of the concentration of detergent (CHAPS) up to 10 mM. PMID:2809063

  17. Isolation of pregnancy-associated glycoproteins (PAG) from water buffalo (Bubalus bubalis) placenta by use of Vicia villosa bound agarose affinity chromatography

    OpenAIRE

    Beckers, J. F.; Malfatti, A.; V. Barile; Debenedetti, A.; Clerget, E.; K. Klisch; Sousa, N.M.; O. Barbato

    2010-01-01

    The present study describes the isolation and characterisation of new PAG molecules extracted from mid- and late-pregnancy placentas in the water buffalo (Bubalis bubalis). After extraction, acid and ammonium sulphate precipitation and DEAE chromatography water buffalo PAG (wbPAG) were enriched by Vicia villosa agarose (VVA) affininity chromatography. As determined by Western blotting with anti-PAG-sera, apparent molecular masses of immunoreactive bands from VVA peaks ranged from 59.5 to 75.8...

  18. A tailored three-dimensionally printable agarose-collagen blend allows encapsulation, spreading, and attachment of human umbilical artery smooth muscle cells.

    Science.gov (United States)

    Köpf, Marius; Campos, Daniela F Duarte; Blaeser, Andreas; Sen, Kshama S; Fischer, Horst

    2016-01-01

    In recent years, novel biofabrication technologies have enabled the rapid manufacture of hydrogel-cell suspensions into tissue-imitating constructs. The development of novel materials for biofabrication still remains a challenge due to a gap between contradicting requirements such as three-dimensional printability and optimal cytocompatibility. We hypothesise that blending of different hydrogels could lead to a novel material with favourable biological and printing properties. In our work, we combined agarose and type I collagen in order to develop a hydrogel blend capable of long-term cell encapsulation of human umbilical artery smooth muscle cells (HUASMCs) and 3D drop-on-demand printing. Different blends were prepared with 0.25%, 0.5%, 0.75%, and 1.5% agarose and 0.2% type I collagen. The cell morphology of HUASMCs and the printing accuracy were assessed for each agarose-collagen combination, keeping the content of collagen constant. The hydrogel blend which displayed sufficient cell spreading and printing accuracy (0.5% agarose, 0.2% type I collagen, AGR0.5COLL0.2) was then characterised based on swelling and degradation over 21 days and mechanical stiffness. The cellular response regarding cell attachment of HUASMCs embedded in the hydrogel blend was further studied using SEM, TEM, and TPLSM. Printing trials were fabricated in a drop-on-demand printing process. The swelling and degradation evaluation showed an average of 20% mass loss and less than 10% swelling. AGR0.5COLL0.2 exhibited significant increase in stiffness compared to pure agarose and type I collagen. In addition, columns of AGR0.5COLL0.2 three centimeters in height were successfully printed submerged in cooled perfluorocarbon, proving the intrinsic printability of the hydrogel blend. Ultimately, a promising novel hydrogel blend showing cell spreading and attachment as well as suitability for bioprinting was identified and could, for example, serve in the manufacture of in vitro 3D models to

  19. Evaluation of Three-Dimensional Chitosan-Agarose-Gelatin Cryogel Scaffold for the Repair of Subchondral Cartilage Defects: An In Vivo Study in a Rabbit Model

    OpenAIRE

    Gupta, Ankur; Bhat, Sumrita; Jagdale, Pankaj R.; Bhushan P Chaudhari; Lidgren, Lars; Gupta, Kailash C.; Kumar, Ashok

    2014-01-01

    In this study, the potential of a chitosan-agarose-gelatin (CAG) cryogel scaffold for the repair of subchondral cartilage defects was explored in female New Zealand white rabbits. Custom-made CAG cryogel scaffold was implanted in a surgically created subchondral defect (diameter of 4 mm, depth of 4 mm) in knee joint of rabbit. The repair of the subchondral defect was evaluated at regular time interval by both macroscopic as well as microscopic examinations. The gross evaluation of the scaffol...

  20. A subtle calculation method for nanoparticle’s molar extinction coefficient: The gift from discrete protein-nanoparticle system on agarose gel electrophoresis

    Science.gov (United States)

    Zhong, Ruibo; Yuan, Ming; Gao, Haiyang; Bai, Zhijun; Guo, Jun; Zhao, Xinmin; Zhang, Feng

    2016-03-01

    Discrete biomolecule-nanoparticle (NP) conjugates play paramount roles in nanofabrication, in which the key is to get the precise molar extinction coefficient of NPs. By making best use of the gift from a specific separation phenomenon of agarose gel electrophoresis (GE), amphiphilic polymer coated NP with exact number of bovine serum albumin (BSA) proteins can be extracted and further experimentally employed to precisely calculate the molar extinction coefficient of the NPs. This method could further benefit the evaluation and extraction of any other dual-component NP-containing bio-conjugates.

  1. Measurement of the ferric diffusion coefficient in agarose and gelatine gels by utilization of the evolution of a radiation induced edge as reflected in relaxation rate images

    International Nuclear Information System (INIS)

    A method has been developed to determine the diffusion coefficients of ferric ions in ferrous sulphate doped gels. A radiation induced edge was created in the gel, and two spin-echo sequences were used to acquire a pair of images of the gel at different points of time. For each of these image pairs, a longitudinal relaxation rate image was derived. From profiles through these images, the standard deviations of the Gaussian functions that characterize diffusion were determined. These data provided the basis for the determination of the ferric diffusion coefficients by two different methods. Simulations indicate that the use of single spin-echo images in this procedure may in some cases lead to a significant underestimation of the diffusion coefficient. The technique was applied to different agarose and gelatine gels that were prepared, irradiated and imaged simultaneously. The results indicate that the diffusion coefficient is lower in a gelatine gel than in an agarose gel. Addition of xylenol orange to a gelatine gel lowers the diffusion coefficient from 1.45 to 0.81 mm2 h-1, at the cost of significantly lower R1 sensitivity. The addition of benzoic acid to the latter gel did not increase the R1 sensitivity. (author) OK

  2. Detection of bacteriophage phi 6 minus-strand RNA and novel mRNA isoconformers synthesized in vivo and in vitro, by strand-separating agarose gels

    International Nuclear Information System (INIS)

    Two urea-free agarose gel protocols that resolve the six individual strands of bacteriophage phi 6 dsRNA were developed and used to analyze phage RNA synthesis in vivo and in vitro. Citrate gels separate strands of the large and medium chromosomes while Tris-borate-EDTA (TBE) gels resolve the medium and small dsRNA segments. Minus strands migrate faster than plus strands on citrate gels but are retarded on TBE gels. A study of electrophoretic conditions showed that pH affects strand resolution on citrate gels, and that voltage gradient, agarose concentration, and ethidium bromide significantly alter strand migration on TBE gels. Analysis of native phi 6 RNA synthesized in vivo and in vitro showed that the large and medium message RNAs comigrate with the corresponding plus strands of denatured virion dsRNA. The small messenger RNA is exceptional. Native small mRNA was detected as three isoconformers in vivo and in vitro. The isoconformers were converted by heat denaturation to a single RNA species that comigrates with the virion s+ strand. Minus strands labeled in vivo were detected only after heat denaturation. Minus strand synthesis was detected also in heat-denatured samples from in vitro phi 6 nucleocapsid RNA polymerase reactions at pH values suboptimal for transcription

  3. Facile preparation of agarose-chitosan hybrid materials and nanocomposite ionogels using an ionic liquid via dissolution, regeneration and sol-gel transition

    CERN Document Server

    Trivedi, Tushar J; Kumar, Arvind

    2014-01-01

    We report simultaneous dissolution of agarose (AG) and chitosan (CH) in varying proportions in an ionic liquid (IL), 1-butyl-3-methylimidazolium chloride [C4mim][Cl]. Composite materials were constructed from AG-CH-IL solutions using the antisolvent methanol, and IL was recovered from the solutions. Composite materials could be uniformly decorated with silver oxide (Ag2O) nanoparticles (Ag NPs) to form nanocomposites in a single step by in situ synthesis of Ag NPs in AG-CH-IL sols, wherein the biopolymer moiety acted as both reducing and stabilizing agent. Cooling of Ag NPs-AG-CH-IL sols to room temperature resulted in high conductivity and high mechanical strength nanocomposite ionogels. The structure, stability and physiochemical properties of composite materials and nanocomposites were characterized by several analytical techniques, such as Fourier transform infrared (FTIR), CD spectroscopy, differential scanning colorimetric (DSC), thermogravimetric analysis (TGA), gel permeation chromatography (GPC), and...

  4. Radiotherapeutic response of Ehrlich Ascites tumor cells perfused in agarose gel threads and implanted in mice. A 31P MR spectroscopy study

    International Nuclear Information System (INIS)

    Aim: In order to obtain better understanding of radiation-induced alterations in intracellular metabolism, a dynamic and noninvasive experimental model system is required. A serial study in cultured tumor cell line followed by verification in the in vivo samples may be of considerable value for non-invasive prediction and/or detection of tumor response to therapy. The present study was undertaken to evaluate the radiation response of perfused Ehrlich ascites tumors cells (EATC) immobilized in agarose gel matrix to that observed in mouse bearing EATC tumor, in order to identify biomarkers of radiation response. Materials and Methods: Perfused EAT cells, entrapped in agarose gel threads were irradiated in the perfusion assembly outside the magnet with fast electrons (6 Gy, 1 Gy/min) using 30 MeV Betatron. Solid EATC tumors implanted subcutaneously onto right hand limb of Swiss-albino strain 'A' mice, were focally irradiated using 60Co teletherapy (10 Gy, 0.4 Gy/min). Metabolites changes were monitored by 31P MR spectroscopic techniques. Results: A post-irradiation decrease in the levels of ATP and ADP along with an increase in inorganic phosphate and glycerophosphocholine levels was observed. The ratios of β-phosphate of ATP to inorganic phosphate (β-ATP-Pi), and phosphocholine to glycerophosphocholine (PC/GPC), declined during 1-5 hours following irradiation, in perfused EAT cells and in the solid tumors implanted in mice. Conclusion: Perfused cells could be used as a simple model of tumor for prediction of clinical radiotherapeutic response. The present study demonstrates that radiation damage may be occurring both at the DNA protein as well as the membrane lipid levels. Therefore, the bioenergetics and phospholipid profiles of tumor cells could be used as complimentary, reliable and sensitive indirect indicators for devising predictive assays for assessment and monitoring of radiation response, which will also facilitate the individualization and optimization of

  5. Isolation of pregnancy-associated glycoproteins (PAG from water buffalo (Bubalus bubalis placenta by use of Vicia villosa bound agarose affinity chromatography

    Directory of Open Access Journals (Sweden)

    J.F. Beckers

    2010-02-01

    Full Text Available The present study describes the isolation and characterisation of new PAG molecules extracted from mid- and late-pregnancy placentas in the water buffalo (Bubalis bubalis. After extraction, acid and ammonium sulphate precipitation and DEAE chromatography water buffalo PAG (wbPAG were enriched by Vicia villosa agarose (VVA affininity chromatography. As determined by Western blotting with anti-PAG-sera, apparent molecular masses of immunoreactive bands from VVA peaks ranged from 59.5 to 75.8 kDa and from 57.8 to 80.9 kDa in the mid- and late- pregnancy placenta respectively. Aminoterminal microsequencing of proteins allowed the identification of three distinct wbPAG sequences wich have ben deposed in the SwissProt database: RGSXLTIHPLRNIRDFFYUG (Acc. n. P85048, RGSXLTILPLRNIID (P85049 and RGSXLTHLPLRNI (P85050. Their comparison to those previously identified revealed that two of them were new since they have not been described yet. Our results confirm the suitability of VVA chromatography in enrichment of multiple PAG molecules expressed in buffalo placenta. Productions of specific antisera can be very useful in immonoistochemical and immunocyitochemical studies of PAG expression in fetomaternal interfaces. Purified native PAG are also required for development on specific immoassays (RIA/ELISA currently used for pregnancy diagnosis and physiological investigation in farm animal.

  6. Identification of column edges of DNA fragments by using K-means clustering and mean algorithm on lane histograms of DNA agarose gel electrophoresis images

    Science.gov (United States)

    Turan, Muhammed K.; Sehirli, Eftal; Elen, Abdullah; Karas, Ismail R.

    2015-07-01

    Gel electrophoresis (GE) is one of the most used method to separate DNA, RNA, protein molecules according to size, weight and quantity parameters in many areas such as genetics, molecular biology, biochemistry, microbiology. The main way to separate each molecule is to find borders of each molecule fragment. This paper presents a software application that show columns edges of DNA fragments in 3 steps. In the first step the application obtains lane histograms of agarose gel electrophoresis images by doing projection based on x-axis. In the second step, it utilizes k-means clustering algorithm to classify point values of lane histogram such as left side values, right side values and undesired values. In the third step, column edges of DNA fragments is shown by using mean algorithm and mathematical processes to separate DNA fragments from the background in a fully automated way. In addition to this, the application presents locations of DNA fragments and how many DNA fragments exist on images captured by a scientific camera.

  7. Characterization of β-Glucosidase Produced by Aspergillus niger under Solid-State Fermentation and Partially Purified Using MANAE-Agarose

    Directory of Open Access Journals (Sweden)

    Anderson Baraldo Junior

    2014-01-01

    Full Text Available β-Glucosidase (BGL is a hydrolytic enzyme with specificity for a wide variety of glycoside substrates, being an enzyme with a large range of biotechnological applications. However, enzyme properties can be different depending both on the microorganism and the cultivation procedure employed. Therefore, in order to explore potential biocatalytical applications of novel enzymes, their characterization is essential. In this work, a BGL synthesized by a selected strain of Aspergillus niger cultivated under solid-state fermentation (SSF was partially purified and fully characterized in terms of optimum pH, temperature, and thermostability. The single-step purification using MANAE-agarose in a chromatographic column yielded an enzyme solution with specific activity (17.1 IU/mg protein adequate for the characterization procedures. Electrophoresis SDS-PAGE and size-exclusion chromatography analysis resulted in an estimated molecular mass of 60 kDa. Higher enzyme activities were found in the range between 40 and 65°C and between pH 4 and 5.5, indicating an interesting characteristic for application in the hydrolysis of lignocellulosic biomass for biofuels production. Thermostability studies of purified BGL resulted in half-lives at 37°C of 56.3 h and at 50°C of 5.4 h. These results provide support for further studies of this enzyme towards revealing its potential biotechnological applications.

  8. Highly selective and sensitive optical sensor for determination of Pb2+and Hg2+ ions based on the covalent immobilization of dithizone on agarose membrane

    Science.gov (United States)

    Zargoosh, Kiomars; Babadi, Fatemeh Farhadian

    2015-02-01

    A highly sensitive and selective optical membrane for determination of Hg2+ and Pb2+ was prepared by covalent immobilization of dithizone on agarose membrane. In addition to its high stability, reproducibility and relatively long lifetime, the proposed optical sensor revealed good selectivity for target ions over a large number of alkali, alkaline earth, transition, and heavy metal ions. The proposed optical membrane displays linear responses from 1.1 × 10-8 to 2.0 × 10-6 mol L-1 and 1.2 × 10-8 to 2.4 × 10-6 mol L-1 for Hg2+ and Pb2+, respectively. The limits of detection (LOD) were 2.0 × 10-9 mol L-1 and 4.0 × 10-9 mol L-1 for Hg2+ and Pb2, respectively. The prepared optical membrane was successfully applied to the determination of Hg2+ and Pb2+ in industrial wastes, spiked tap water and natural waters without any preconcentration step.

  9. Anaerobic crystallization and initial X-ray diffraction data of biphenyl 2,3-dioxygenase from Burkholderia xenovorans LB400: addition of agarose improved the quality of the crystals

    International Nuclear Information System (INIS)

    Biphenyl 2,3-dioxygenase from B. xenovorans LB400 and its variants BPDOP4 and BPDORR41 were crystallized using agarose gel and the crystals were characterized using X-ray diffraction. Biphenyl 2,3-dioxygenase (BPDO; EC 1.14.12.18) catalyzes the initial step in the degradation of biphenyl and some polychlorinated biphenyls (PCBs). BPDOLB400, the terminal dioxygenase component from Burkholderia xenovorans LB400, a proteobacterial species that degrades a broad range of PCBs, has been crystallized under anaerobic conditions by sitting-drop vapour diffusion. Initial crystals obtained using various polyethylene glycols as precipitating agents diffracted to very low resolution (∼8 Å) and the recorded reflections were diffuse and poorly shaped. The quality of the crystals was significantly improved by the addition of 0.2% agarose to the crystallization cocktail. In the presence of agarose, wild-type BPDOLB400 crystals that diffracted to 2.4 Å resolution grew in space group P1. Crystals of the BPDOP4 and BPDORR41 variants of BPDOLB400 grew in space group P21

  10. Imaging of VSOP labeled stem cells in agarose phantoms with susceptibility weighted and T2* weighted MR Imaging at 3T: determination of the detection limit.

    Directory of Open Access Journals (Sweden)

    Donald Lobsien

    Full Text Available OBJECTIVES: This study aimed to evaluate the detectability of stem cells labeled with very small iron oxide particles (VSOP at 3T with susceptibility weighted (SWI and T2* weighted imaging as a methodological basis for subsequent examinations in a large animal stroke model (sheep. MATERIALS AND METHODS: We examined ovine mesenchymal stem cells labeled with VSOP in agarose layer phantoms. The experiments were performed in 2 different groups, with quantities of 0-100,000 labeled cells per layer. 15 different SWI- and T2*-weighted sequences and 3 RF coils were used. All measurements were carried out on a clinical 3T MRI. Images of Group A were analyzed by four radiologists blinded for the number of cells, and rated for detectability according to a four-step scale. Images of Group B were subject to a ROI-based analysis of signal intensities. Signal deviations of more than the 0.95 confidence interval in cell containing layers as compared to the mean of the signal intensity of non cell bearing layers were considered significant. RESULTS: GROUP A: 500 or more labeled cells were judged as confidently visible when examined with a SWI-sequence with 0.15 mm slice thickness. Group B: 500 or more labeled cells showed a significant signal reduction in SWI sequences with a slice thickness of 0.25 mm. Slice thickness and cell number per layer had a significant influence on the amount of detected signal reduction. CONCLUSION: 500 VSOP labeled stem cells could be detected with SWI imaging at 3 Tesla using an experimental design suitable for large animal models.

  11. Application of urea-agarose gel electrophoresis to select non-redundant 16S rRNAs for taxonomic studies: palladium(II) removal bacteria.

    Science.gov (United States)

    Assunção, Ana; Costa, Maria Clara; Carlier, Jorge Dias

    2016-03-01

    The 16S ribosomal RNA (rRNA) gene has been the most commonly used sequence to characterize bacterial communities. The classical approach to obtain gene sequences to study bacterial diversity implies cloning amplicons, selecting clones, and Sanger sequencing cloned fragments. A more recent approach is direct sequencing of millions of genes using massive parallel technologies, allowing a large-scale biodiversity analysis of many samples simultaneously. However, currently, this technique is still expensive when applied to few samples; therefore, the classical approach is still used. Recently, we found a community able to remove 50 mg/L Pd(II). In this work, aiming to identify the bacteria potentially involved in Pd(II) removal, the separation of urea/heat-denatured DNA fragments by urea-agarose gel electrophoresis was applied for the first time to select 16S rRNA-cloned amplicons for taxonomic studies. The major raise in the percentage of bacteria belonging to genus Clostridium sensu stricto from undetected to 21 and 41 %, respectively, for cultures without, with 5 and 50 mg/L Pd(II) accompanying Pd(II) removal point to this taxa as a potential key agent for the bio-recovery of this metal. Despite sulfate-reducing bacteria were not detected, the hypothesis of Pd(II) removal by activity of these bacteria cannot be ruled out because a slight decrease of sulfate concentration of the medium was verified and the formation of PbS precipitates seems to occur. This work also contributes with knowledge about suitable partial 16S rRNA gene regions for taxonomic studies and shows that unidirectional sequencing is enough when Sanger sequencing cloned 16S rRNA genes for taxonomic studies to genus level. PMID:26590590

  12. Emprego do cell block de agarose como método complementar no diagnóstico citológico de tumores mamários caninos Employment of cell block of agarose as additional method in the cytological diagnosis of canine mammary tumors

    Directory of Open Access Journals (Sweden)

    Diogo Sousa Zanoni

    2013-03-01

    Full Text Available Os tumores mamários são neoplasias comuns em diversas espécies, sendo os processos oncológicos de maior incidência em cães. A elevada frequência e agressividade desses processos justificam a busca de métodos diagnósticos e prognósticos rápidos, de custo reduzido e menor invasividade, visando a uma abordagem cirúrgica e terapêutica adequada. O presente estudo avaliou a adequação da utilização da técnica de cell block de agarose como método diagnóstico complementar aos esfregaços tradicionais no diagnóstico desses processos. Para tanto, foram obtidas 51 amostras citológicas de tumores mamários de 30 cadelas que passaram por excisão tumoral no HOVET-UMESP, comparando-se os resultados obtidos a partir dos esfregaços, de cell blocks, e de sua associação (esfregaços cell blocks-1 com o diagnóstico histopatológico. Os melhores resultados foram obtidos mediante a associação dos métodos, reduzindo os resultados falso-negativos e elevando a correlação cito-histológica, reforçando a importância da citologia na rotina oncológica veterinária.The breast tumors are common neoplasms in several species, with high incidence in dogs. The high frequency and aggressiveness of these cases justifies the search for rapid, low cost and less invasive diagnostic methods, seeking for surgical approach and appropriate therapy. This study evaluated the appropriateness of the use of the agarose cell block technique as a diagnostic tool to complement traditional smears in the diagnosis of these processes. Therefore, it was obtained 51 samples from 30 dogs with breast tumors that underwent tumoral excision at the HOVET-UMESP, comparing the results obtained from smears, cell blocks, alone and in association (smears cell blocks-1, with the histopathologic diagnosis. The best results were obtained with the association of smears and cell block analysis, reducing the false negative results and increasing the cyto-histological correlation

  13. Integrated system for temperature-controlled fast protein liquid chromatography comprising improved copolymer modified beaded agarose adsorbents and a travelling cooling zone reactor arrangement.

    Science.gov (United States)

    Müller, Tobias K H; Cao, Ping; Ewert, Stephanie; Wohlgemuth, Jonas; Liu, Haiyang; Willett, Thomas C; Theodosiou, Eirini; Thomas, Owen R T; Franzreb, Matthias

    2013-04-12

    An integrated approach to temperature-controlled chromatography, involving copolymer modified agarose adsorbents and a novel travelling cooling zone reactor (TCZR) arrangement, is described. Sepharose CL6B was transformed into a thermoresponsive cation exchange adsorbent (thermoCEX) in four synthetic steps: (i) epichlorohydrin activation; (ii) amine capping; (iii) 4,4'-azobis(4-cyanovaleric acid) immobilization; and 'graft from' polymerization of poly(N-isopropylacrylamide-co-N-tert-butylacrylamide-co-acrylic acid-co-N,N'-methylenebisacrylamide). FT-IR, (1)H NMR, gravimetry and chemical assays allowed precise determination of the adsorbent's copolymer composition and loading, and identified the initial epoxy activation step as a critical determinant of 'on-support' copolymer loading, and in turn, protein binding performance. In batch binding studies with lactoferrin, thermoCEX's binding affinity and maximum adsorption capacity rose smoothly with temperature increase from 20 to 50 °C. In temperature shifting chromatography experiments employing thermoCEX in thermally jacketed columns, 44-51% of the lactoferrin adsorbed at 42 °C could be desorbed under binding conditions by cooling the column to 22 °C, but the elution peaks exhibited strong tailing. To more fully exploit the potential of thermoresponsive chromatography adsorbents, a new column arrangement, the TCZR, was developed. In TCZR chromatography, a narrow discrete cooling zone (special assembly of copper blocks and Peltier elements) is moved along a bespoke fixed-bed separation columnfilled with stationary phase. In tests with thermoCEX, it was possible to recover 65% of the lactoferrin bound at 35 °C using 8 successive movements of the cooling zone at a velocity of 0.1mm/s; over half of the recovered protein was eluted in the first peak in more concentrated form than in the feed. Intra-particle diffusion of desorbed protein out of the support pores, and the ratio between the velocities of the cooling

  14. 南宁地区琼脂糖凝胶电泳产前筛查地贫16 198例结果分析%Analysis on 16 198 cases prenatal screen of Thalassaemia using agarose electrophoresis

    Institute of Scientific and Technical Information of China (English)

    阙婷; 李东明

    2012-01-01

    目的 评价琼脂糖凝胶电泳在产前地中海贫血(地贫)筛查中的价值.方法 采用琼脂糖凝胶电泳对16 198例外周血或胎儿脐血进行Hb分析,其中4 242例进行地贫基因检测.结果 16 198例检出β地贫1390例,异常Hb 495例,异常Hb病133例,异常检出率12.46%.与基因检测结果比较,电泳筛查β地贫、HbH病和HbCS的灵敏度和特异度为93.57%和96.25%、100%和69.74%及99.64%和62.52%,以HbA2≤2.45%、2.65%筛查α1、α2地贫的灵敏度和特异度分别为68.44%和64.11%、63.81%和52.79%.电泳筛查胎儿脐血α地贫的灵敏度和特异度为89.94%和98.58%,其中对中间型、重型α地贫筛查灵敏度和特异性均为100%.结论 广西地区地贫发生率较高,琼脂糖凝胶电泳筛查β地贫的灵敏度和特异度较好,能准确的筛出胎儿脐血中间型、重型α地贫;在胎儿脐血和外周血中筛查轻型和静止型α地贫的灵敏度和特异度差异较大,且胎儿脐血优于外周血.%Objective: To evaluate the application of agarose electrophoresis in the prenatal screen of thalassemia (thal). Methods: Agarose electrophoresis was used to perform hemoglobin (Hb) electrophoresis for 16 918 cases. 4 242 samples were further examined by DNA analysis. Results; Among 16 198 cases, 1 390 cases of (3 thal, 4S9 cases of abnormal hemoglobin and 133 cases of abnormal hemoglobin disease were detected, and the abnormal rate was 12. 46% . The sensitivity and specificity that agarose electrophoresis consistent with gene diagnoses for β - thal, HbH disease and HbCS were 93. 57% and 96. 25% , 100% and 69. 74% , 99. 64% and 52. 79%. To diagnose α thal 1 and α thal 2 by HbA2≤2. 45% and 2. 65% , the sensitivity and specificity were 68. 44% and 64. 1 J% , 63. 81% and 52. 79%. The sensitivity and specificity of screening α thai in prenatal umbilical cord blood were 89. 94% and 98. 58% , and the sensitivity and specificity of agarose electrophoresis

  15. In situ selective determination of methylmercury in river water by diffusive gradient in thin films technique (DGT) using baker's yeast (Saccharomyces cerevisiae) immobilized in agarose gel as binding phase

    Energy Technology Data Exchange (ETDEWEB)

    Tafurt-Cardona, Makenly [Programa de Pós-graduação em Geociências e Meio Ambiente, Instituto de Geociências e Ciências Exatas, UNESP – Univ. Estadual Paulista, Av. 24-A, 1515, CEP: 13506-900, Rio Claro, SP (Brazil); Centro de Estudos Ambientais, UNESP – Univ. Estadual Paulista, Av. 24-A, 1515, CEP: 13506-900, Rio Claro, SP (Brazil); Eismann, Carlos Eduardo; Suárez, Carlos Alfredo [Centro de Estudos Ambientais, UNESP – Univ. Estadual Paulista, Av. 24-A, 1515, CEP: 13506-900, Rio Claro, SP (Brazil); Menegário, Amauri Antonio, E-mail: amenega@rc.unesp.br [Programa de Pós-graduação em Geociências e Meio Ambiente, Instituto de Geociências e Ciências Exatas, UNESP – Univ. Estadual Paulista, Av. 24-A, 1515, CEP: 13506-900, Rio Claro, SP (Brazil); Centro de Estudos Ambientais, UNESP – Univ. Estadual Paulista, Av. 24-A, 1515, CEP: 13506-900, Rio Claro, SP (Brazil); Silva Luko, Karen [Programa de Pós-graduação em Geociências e Meio Ambiente, Instituto de Geociências e Ciências Exatas, UNESP – Univ. Estadual Paulista, Av. 24-A, 1515, CEP: 13506-900, Rio Claro, SP (Brazil); Centro de Estudos Ambientais, UNESP – Univ. Estadual Paulista, Av. 24-A, 1515, CEP: 13506-900, Rio Claro, SP (Brazil); and others

    2015-08-05

    Saccharomyces cerevisiae immobilized in agarose gel as binding phase and polyacrylamide as diffusive layer in the diffusive gradient in thin films technique (DGT) was used for selective determination of methylmercury (MeHg). Deployment tests showed good linearity in mass uptake up to 48 h (3276 ng). When coupling the DGT technique with Cold Vapor Atomic Fluorescence Spectrometry, the method has a limit of detection of 0.44 ng L{sup −1} (pre concentration factor of 11 for 48 h deployment). Diffusion coefficient of 7.03 ± 0.77 × 10{sup −6} cm{sup 2} s{sup −1} at 23 °C in polyacrylamide gel (pH = 5.5 and ionic strength = 0.05 mol L{sup −1} NaCl) was obtained. Influence of ionic strength (from 0.0005 mol L{sup −1} to 0.1 mol L{sup −1} NaCl) and pH (from 3.5 to 8.5) on MeHg uptake were evaluated. For these range, recoveries of 84–105% and 84–98% were obtained for ionic strength and pH respectively. Potential interference due to presence of Cu, Fe, Mn, Zn was also assessed showing good recoveries (70–87%). The selectivity of the proposed approach was tested by deployments in solutions containing MeHg and Hg(II). Results obtained showed recoveries of 102–115 % for MeHg, while the uptake of Hg(II) was insignificant. The proposed approach was successfully employed for in situ measurements in the Negro River (Manaus-AM, Brazil). - Highlights: • A method for in situ selective determination of MeHg by DGT technique is proposed. • Saccharomyces cerevisiae immobilized in agarose gel was used as binding agent. • Effects of pH, ionic strength and concomitant ions on uptake of MeHg were evaluated. • DGT device containing polyacrylamide gel as diffusive layer showed better selectivity. • The proposed approach was successfully applied for analysis of river water.

  16. In situ selective determination of methylmercury in river water by diffusive gradient in thin films technique (DGT) using baker's yeast (Saccharomyces cerevisiae) immobilized in agarose gel as binding phase

    International Nuclear Information System (INIS)

    Saccharomyces cerevisiae immobilized in agarose gel as binding phase and polyacrylamide as diffusive layer in the diffusive gradient in thin films technique (DGT) was used for selective determination of methylmercury (MeHg). Deployment tests showed good linearity in mass uptake up to 48 h (3276 ng). When coupling the DGT technique with Cold Vapor Atomic Fluorescence Spectrometry, the method has a limit of detection of 0.44 ng L−1 (pre concentration factor of 11 for 48 h deployment). Diffusion coefficient of 7.03 ± 0.77 × 10−6 cm2 s−1 at 23 °C in polyacrylamide gel (pH = 5.5 and ionic strength = 0.05 mol L−1 NaCl) was obtained. Influence of ionic strength (from 0.0005 mol L−1 to 0.1 mol L−1 NaCl) and pH (from 3.5 to 8.5) on MeHg uptake were evaluated. For these range, recoveries of 84–105% and 84–98% were obtained for ionic strength and pH respectively. Potential interference due to presence of Cu, Fe, Mn, Zn was also assessed showing good recoveries (70–87%). The selectivity of the proposed approach was tested by deployments in solutions containing MeHg and Hg(II). Results obtained showed recoveries of 102–115 % for MeHg, while the uptake of Hg(II) was insignificant. The proposed approach was successfully employed for in situ measurements in the Negro River (Manaus-AM, Brazil). - Highlights: • A method for in situ selective determination of MeHg by DGT technique is proposed. • Saccharomyces cerevisiae immobilized in agarose gel was used as binding agent. • Effects of pH, ionic strength and concomitant ions on uptake of MeHg were evaluated. • DGT device containing polyacrylamide gel as diffusive layer showed better selectivity. • The proposed approach was successfully applied for analysis of river water

  17. Thermoresponsive Agarose Based Microparticles for Antibody Separation.

    Science.gov (United States)

    Ooi, Huey Wen; Ketterer, Benedikt; Trouillet, Vanessa; Franzreb, Matthias; Barner-Kowollik, Christopher

    2016-01-11

    We report the development of thermoresponsive 4-mercaptoethylpyridine (MEP)-based chromatographic microsphere based resins for antibody separation that show switchable release abilities by adsorbing immunoglobulins at 40 °C and releasing the proteins at 5 °C. The thermoswitchable release properties were introduced to the porous resins by the grafting of linear poly(N-isopropylacrylamide) (PNIPAM) chains synthesized via reversible addition-fragmentation chain transfer (RAFT) polymerization, which were modified to possess MEP end functionalities. Adsorption of γ-globulins as a model antibody on the shortest PNIPAM-MEP (3 kDa) grafted microparticles display binding capacities of up to 20 g L(-1) at 40 °C and a significant decrease in binding capacity to less than 2.5 g L(-1) at 5 °C. By switching the temperature to 5 °C, the release of bound γ-globulins is shown to be as high as 90%. The effects of polymer chain length on the binding capacity are studied in detail and found to be critical as they influence the density of MEP functionalities on the particle surfaces. PMID:26626821

  18. 琼脂糖凝胶电泳法分离金属性和半导体性单壁碳纳米管%Separation of Metallic Single-walled Carbon Nanotubes and Semiconducting Single-walled Carbon Nanotubes by Agarose Gel Electrophoresis

    Institute of Scientific and Technical Information of China (English)

    朱胜男; 张静; 李清文; 李红波; 金赫华; 宋启军

    2012-01-01

    The agarose gel electrophoresis (AGE) is one of the low-cost, large scale technologies for the separation of metallic single-walled carbon nanotubes (m-SWCNTs) and semiconducting single-walled carbon nanotubes(s-SWCNTs). The separated m-SWCNTs are divided into several parts and characterized by the UV-visible-near infrared absorption spectrum and the Raman spectrum respectively. The results show that the moieties with the fastest electrophoresis migration rate contain more m-SWCNTs. Furthermore, the effects of different agarose concentrations on the separating efficiencies of SWCNTs are investigated. It is found that higher concentration of agarose gel is beneficial to the enrichment of the m-SWCNTs and the separating efficiency of the m-SWCNTs could be realized by increasing the charge density on the surface of the SWCNTs.%琼脂糖凝胶电泳(AGE)是实现金属性单壁碳纳米管(m-SWCNTs)和半导体性单壁碳纳米管(s-SWCNTs)低成本、规模化分离的有效技术之一.本研究利用琼脂糖凝胶电泳分离单壁碳纳米管.通过紫外-可见-近红外吸收光谱和拉曼光谱对色谱带进行分段表征,发现电泳中迁移的最快的部分m-SWCNTs含量最高.考察了琼脂糖的浓度对SWCNTs中m-SWCNTs分离的影响.结果表明:高的琼脂糖浓度有利于m-SWCNTs的富集,可以通过扩大电荷密度带来的迁移速率的差异来使SWCNTs中的m-SWCNTs得到更有效的分离.

  19. 琼脂糖性质对凝胶电泳法分离金属性和半导体性单壁碳纳米管的影响%Effect of Agarose Properties on the Separation of m-SWCNTs and s-SWCNTs by the AGE

    Institute of Scientific and Technical Information of China (English)

    张静; 温晓南; 李红波; 金赫华; 宋启军; 李清文

    2010-01-01

    利用琼脂糖凝胶电泳分离单壁碳纳米管(SWCNTs)技术, 考察了MB, Agarose, Agarose B和LRU 4种琼脂糖对SWCNTs分离效率的影响. 紫外-可见-近红外(UV-Vis-NIR)吸收光谱研究结果表明, 不同的琼脂糖对SWCNTs中s-SWCNTs的分离效率影响较小, 而对m-SWCNTs的分离效率影响较大. 分析4种琼脂糖凝胶的凝胶强度和凝胶网孔尺寸等发现, 影响SWCNTs中m-SWCNTs分离效率的主要因素是琼脂糖的凝胶强度和琼脂糖凝胶形成的网孔尺寸, 小的凝胶网孔尺寸有利于m-SWCNTs富集, 高凝胶强度则不利于其富集.

  20. Caracterización de la diversidad genética en naranja y comparación del polimorfismo de microsatélites amplificados al azar (RAMs usando electroforesis de poliacrilamida y azarosa Characterization of the genetic diversity in orange, and comparison of polymorphism in randomly-amplifed microsatellites (RAMs, using polyacrylamide and agarose electrophoresis

    Directory of Open Access Journals (Sweden)

    Ana Cruz Morillo Coronado

    2009-10-01

    Full Text Available Se compararon las eficiencias de tres métodos de electroforesis en agarosa y poliacrilamida, usando la cámara pequeña de DNA Sequencing System y cámara grande OWL Sequi-Gen Sequencing Cell, en la detección del polimorfismo en 21 accesiones de naranja (Citrus sinensis con empleo del cebador CGA. El gel de poliacrilamida dio mejor resolución de los productos amplificados vía PCR producidos por RAMs. Este permitió una mejor detección de bandas de ADN polimórficas, lo que facilitó la identificación de la variabilidad genética. La electroforesis en agarosa puede ser más conveniente en otras aplicaciones, debido al bajo costo y fácil aplicación. El estudio de diversidad genética en naranja usando microsatélites RAMs diferenció 51 accesiones en siete grupos con 0.75 de similaridad y 0.25 de heterocigosidad, lo que revela bajo polimorfismo genético. La técnica RAMs permitió agrupar las accesiones en Comunes o Blancas, Navel y Pigmentadas o Sanguinas.We compared the efficiency of three methods of agarose and polyacrylamide electrophoresis (using the small tank of the DNA Sequencing System and the large OWL Sequi-Gen Sequencing Cell, for the detection of polymorphism in 21 accessions of orange (Citrus sinensis, using the primer CGA. The polyacrylamide gel gave better resolution of the PCR-amplified RAM products. This method allowed better detection of polymorphic DNA bands, facilitating the identification of genetic variability. The agarose electrophoresis may be more convenient in other applications, due to its low cost and easy implementation. The study of genetic diversity in orange using RAMs separated 51 accessions into seven groups with 0.75 similarity, and 0.25 heterozygosity, revealing low genetic polymorphism. The RAMs technique grouped the accessions into “Common or White”, “Navel” and “Pigmented or “Sanguine”.

  1. The Influence of Conditioning Agent on Phosphate Diffusion Coefficient through Polyacrylamide and Agarose Gel

    OpenAIRE

    Layta Dinira; Barlah Rumhayati; Rachmat Triandi Tjahjanto

    2013-01-01

    Excess phosphate in natural water can cause algae grow rapidly, to the extent causing many fish deaths that led to the extinction of certain species. Therefore, an analysis or periodic observations of phosphate levels in the water is needed. The commonly used method is diffusive gradient in thin films (DGT) technique. The DGT technique is based on the ability of analyte to diffuse through a gel, which have a value named diffusion coefficient. This research was conducted in order to study the ...

  2. Separation of closed circular DNA from linear DNA by electrophoresis in two dimensions in agarose gels.

    OpenAIRE

    Oppenheim, A

    1981-01-01

    A method that provides an easy, rapid, and reproducible way for separating closed circular DNA species from linear DNA and nicked circles is described. The method is based on the difference in mobility of form I (supercoiled) DNA and form II (nicked circles), and the differential mobility of relaxed circular DNA in the presence and absence of ethidium bromide (EtdBr). It can be used for detection or for purification of plasmid, episomal, or viral DNA from the bulk of cellular DNA, or from oth...

  3. Isolation of DNA fragments containing replication forks by two dimensional agarose gel electrophoresis

    International Nuclear Information System (INIS)

    Escherichia coli is used as a model system to study DNA repair processes that promote survival after damage from alkylating agents and other processes that affect mutation resulting from such damage. In order to test the hypothesis that the induced incision repair process is able to repair this damage, a two-dimensional gel system has been developed that allows resolution of DNA fragments containing replication forks from the rest of the genome

  4. Beverage-Agarose Gel Electrophoresis: An Inquiry-based Laboratory Exercise with Virtual Adaptation1

    OpenAIRE

    Cunningham, Steven C.; McNear, Brad; Rebecca S. Pearlman; Kern, Scott E.

    2006-01-01

    A wide range of literature and experience has shown that teaching methods that promote active learning, such as inquiry-based approaches, are more effective than those that rely on passive learning. Gel electrophoresis, one of the most common laboratory techniques in molecular biology, has a wide range of applications in the life sciences. As such, we chose it as a platform to expose high school and undergraduate students to the active process of scientific inquiry in general, while specifica...

  5. Beverage-Agarose Gel Electrophoresis: An Inquiry-Based Laboratory Exercise with Virtual Adaptation

    Science.gov (United States)

    Cunningham, Steven C.; McNear, Brad; Pearlman, Rebecca S.; Kern, Scott E.

    2006-01-01

    A wide range of literature and experience has shown that teaching methods that promote active learning, such as inquiry-based approaches, are more effective than those that rely on passive learning. Gel electrophoresis, one of the most common laboratory techniques in molecular biology, has a wide range of applications in the life sciences. As…

  6. Minisatellite isoalleles can be distinguished by single-stranded conformational polymorphism analysis in agarose gels.

    OpenAIRE

    Monckton, D.G.; Jeffreys, A J

    1994-01-01

    Minisatellite isoallelism, i.e. the occurrence of minisatellite alleles with different internal sequence composition but indistinguishable length, is a common limitation of minisatellite allele length analysis. Internal sequence variation can be used to distinguish such isoalleles, provided that detailed sequence knowledge of its basis is available. We now show that minisatellite isoalleles can also be simply resolved by single-stranded conformational polymorphisms (SSCP) arising during agaro...

  7. Identification of Cisplatin-Binding Proteins Using Agarose Conjugates of Platinum Compounds

    OpenAIRE

    Takatoshi Karasawa; Martha Sibrian-Vazquez; Strongin, Robert M.; Peter S Steyger

    2013-01-01

    Cisplatin is widely used as an antineoplastic drug, but its ototoxic and nephrotoxic side-effects, as well as the inherent or acquired resistance of some cancers to cisplatin, remain significant clinical problems. Cisplatin's selectivity in killing rapidly proliferating cancer cells is largely dependent on covalent binding to DNA via cisplatin's chloride sites that had been aquated. We hypothesized that cisplatin's toxicity in slowly proliferating or terminally differentiated cells is primari...

  8. Agarose Gel Electrophoresis System in the Classroom: Detection of DNA Strand Breaks through the Alteration of Plasmid Topology

    Science.gov (United States)

    De Mattos, J. C. P.; Dantas, F. J. S.; Caldeira-de-Araujo, A.; Moraes, M. O.

    2004-01-01

    Good quality scientific teaching depends on the ability of researchers to translate laboratory experiments into high school and undergraduate classes, bridging the advanced and basic science with common knowledge. A fast-growing field in biomedical sciences is oxidative stress, which has been associated to several diseases, including cancer and…

  9. Detection and characterization of agarose-binding, capsid-like particles produced during assembly of a bacteriophage T7 procapsid.

    OpenAIRE

    Serwer, P; Watson, R H; Hayes, S J

    1982-01-01

    It has previously been shown that: (i) during infection of its host, the DNA bacteriophage T7 assembles a DNA-free procapsid (capsid I), a capsid with an envelope differing physically and chemically from the capsid of the mature bacteriophage, and (ii) capsid I converts to a capsid (capsid II) with a bacteriophage-like envelope as it packages DNA. Lysates of phage T7-infected Escherichia coli contained a particle (AG particle) which copurified with capsid II during buoyant density sedimentati...

  10. Bacteriophage P22 in vitro DNA packaging monitored by agarose gel electrophoresis: rate of DNA entry into capsids.

    OpenAIRE

    Gope, R.; Serwer, P

    1983-01-01

    Bacteriophage P22, like other double-stranded DNA bacteriophages, packages DNA in a preassembled, DNA-free procapsid. The P22 procapsid and P22 bacteriophage have been electrophoretically characterized; the procapsid has a negative average electrical surface charge density (sigma) higher in magnitude than the negative sigma of the mature bacteriophage. Dextrans, sucrose, and maltose were shown to have a dramatic stimulatory effect on the in vitro packaging of DNA by the P22 procapsid. However...

  11. Agarose cell block technique as a complementary method in the diagnosis of fungal osteomyelitis in a dog

    OpenAIRE

    N.S. Rocha; S.M.G. Bosco; Cagnini, D.Q.; Zanoni, D.S.; Grandi, F

    2012-01-01

    A 7-year-old Labrador Retriever female dog presenting left forelimb lameness for one day was admitted to the Veterinary Hospital (UNESP-Botucatu) for clinical evaluation. Several tests, including blood and image analysis, microbiological culture and cytology of lytic areas of affected bone were made in order to establish a diagnosis. Serum biochemical profile revealed increased levels of liver enzymes, plasma globulin, creatine kinase (CK) and calcium. Hemogram revealed anemia and leukocytosi...

  12. A novel assay for screening inhibitors targeting HIV-1 integrase dimerization based on Ni-NTA magnetic agarose beads.

    Science.gov (United States)

    Zhang, Dawei; He, Hongqiu; Liu, Mengmeng; Meng, Zhixia; Guo, Shunxing

    2016-01-01

    Human immunodeficiency virus (HIV)-1 integrase (IN), which mediates integration of viral cDNA into the cellular chromosome, is a validated antiviral drug target. Three IN inhibitors, raltegravir, elvitegravir and dolutegravir, have been clinically approved since 2008. However, drug resistance have emerged in infected patients receiving treatment using these drugs which share the same mechanism of action and have a low genetic barrier for resistance. Therefore, there is an urgent need to develop drugs with novel mechanism. IN requires a precise and dynamic equilibrium between several oligomeric species for its activities. The modulation of the process which is termed as IN oligomerization, presents an interesting allosteric target for drug development. In this research, we developed a magnetic beads based approach to assay the IN dimerization. Then, using the assay we screened a library of 1000 Food and Drug Administration (FDA)-approved drugs for IN dimerization inhibitors and identified dexlansoprazole as a potential IN dimerization inhibitor. In conclusion, the assay presented here has been proven to be sensitive and specific for the detection of IN dimerization as well as for the identification of antiviral drugs targeting IN dimerization. Moreover, a FDA-approved proton-pump inhibitors, dexlansoprazole, was identified as a potential inhibitor for IN dimerization. PMID:27137477

  13. A charge-coupled-device camera image analysis system for quantifying DNA distributions in agarose gels after pulsed-field gel electrophoresis

    International Nuclear Information System (INIS)

    A charge-coupled-device camera system was coupled to a personal computer and, with uniformity in illumination and detection (within 4-8%) along each lane, was used for quantifying the distribution of DNA molecules that migrate from the PFGE well (plug) into the lane at distances varying from 1 to 50 mm (with 0.5 mm/pixel). By using a specially designed transmission filter for transmitting 470-725 nm fluorescence from ethidium bromide-stained DNA while eliminating most of the fluorescence (3H]dThd. However, scattering of fluorescence from one lane into an adjacent lane 3 mm away and as far as 10 mm from the plug into the lane presented a problem. This problem was overcome by using a form with slots to cover every other lane when the images were obtained and either (1) cutting the lane from the plug and moving it 15 mm away or (2) imaging the intact gel and applying a correction for ∼ 7% of the fluorescence from the plug tailing out ∼ 10 mm beyond the first 1 mm in the lane. In addition, the following were required: (1) carefully controlled staining and destaining procedures, and (2) a low background that is obtained as an average uniform background in each lane 5 mm beyond where DNA migration stops. 31 refs., 7 figs

  14. Estimating the DNA strand breakage using a fuzzy inference system and agarose gel electrophoresis, a case study with toothed carp Aphanius sophiae exposed to cypermethrin.

    Science.gov (United States)

    Poorbagher, Hadi; Moghaddam, Maryam Nasrollahpour; Eagderi, Soheil; Farahmand, Hamid

    2016-07-01

    The DNA breakage has been widely used in ecotoxicological studies to investigate effects of pesticides in fishes. The present study used a fuzzy inference system to quantify the breakage of DNA double strand in Aphanius sophiae exposed to the cypermethrin. The specimens were adapted to different temperatures and salinity for 14 days and then exposed to cypermethrin. DNA of each specimens were extracted, electrophoresed and photographed. A fuzzy system with three input variables and 27 rules were defined. The pixel value curve of DNA on each gel lane was obtained using ImageJ. The DNA breakage was quantified using the pixel value curve and fuzzy system. The defuzzified values were analyzed using a three-way analysis of variance. Cypermethrin had significant effects on DNA breakage. Fuzzy inference systems can be used as a tool to quantify the breakage of double strand DNA. DNA double strand of the gill of A. sophiae is sensitive enough to be used to detect cypermethrin in surface waters in concentrations much lower than those reported in previous studies. PMID:27000282

  15. Results concerning the analysis of the reaction products resulting from genomic dna amplification using agarose gel electrophoresis for potatoes studied old varieties

    Directory of Open Access Journals (Sweden)

    Anca BACIU

    2008-05-01

    Full Text Available The author is currently involved in collecting, making an inventory, evaluation and preservation the old varieties from the Western part of Romania. In this paper 8 potato old varieties collected during 20 years and 2 varieties from National Institute of Research and Development for potato and Sugar Beet Brasov are presented. The preservation was carried out in vivo and in vitro. Important changes were observed during this time. In our work we identified many gaps in the knowledge and understanding of the origin of transformations. We made a comparison between two big areas of potato growth: Apuseni Mountains [5] and the Maramures County [3]. In these areas the potato represents the main food in winter. This work opens opportunities for future researches in the field of political and ethical decisions for potato gene pool conservation. Soon the exchange of genetic resources will be a diplomatic issue.

  16. Determination of G-values for single and double strand break induction in plasmid DNA using agarose gel electrophoresis and a curve-fitting procedure

    International Nuclear Information System (INIS)

    Covalently closed circular double-stranded DNA (CC) of native plasmids was used to determine yields of single strand breaks (ssb) and double strand breaks (dsb) caused by x-radiation. One ssb transforms DNA of the CC form to the nicked circular form (NC); one dsb produced either directly or from random coincidence of single strand breaks transforms DNA of the CC as well as of the NC form to linear DNA molecules (LI form). Plasmids with more than one dsb are cleaved to linear fragments. DNA (30-800 μg/ml) was irradiated in air-saturated buffer. DNA forms were separated by gel electrophoresis and their amounts measured fluorometrically using ethidium bromide. Large linear DNA fragments with the same electrophoretic mobility as the LI form were considered using a curve-fitting procedure. From quantitative changes of each conformation D37 values of ssb and dsb were calculated as a function of the DNA concentration. Finally G-values were calculated by competition plots. The following yields were determined: Gsub(ssb) 3.4 x 10E - 8 mol J-1, and Gsub(dsb) 3.3 x 10E - 10 molJ-1. Gsub(dsb) refers to directly produced dsb. Yields are related to strand breaks without further treatment. (author)

  17. The effects on DNA migration of altering parameters in the comet assay protocol such as agarose density, electrophoresis conditions and durations of the enzyme or the alkaline treatments

    OpenAIRE

    Ersson, C.; MOLLER, L.

    2011-01-01

    The single cell gel electrophoresis (comet assay) is a popular method for measuring DNA migration as an estimate of DNA damage. No standardised comet assay protocol exists, which make comparisons between studies complicated. In a previous inter-laboratory validation study of the comet assay, we identified important parameters in the protocol that might affect DNA migration. The aim of this study was to assess how different comet assay protocols affect DNA migration. The results in this study ...

  18. Agarose gel electrophoresis of joint fluid using Hyrys-Hydrasys SEBIA system as a new prognostic tool for periprosthetic osteolysisin revision arthroplasty

    OpenAIRE

    Chiva, A

    2013-01-01

    Rationale. Prevention of wear-mediated osteolysis, the most common complication in total joint arthroplasty, is a great challenge for orthopedic surgery. Despite the diversity of current biomarkers of periprosthetic osteolysis (products of wear, bone turnover and inflammatory biomarkers), the major interferences and the great amount of sample necessary for analysis limit their use in clinical practice. Objective. The aim of this paper is to present three new electrophoretic methods using Hyry...

  19. Detection of the basement membrane-degrading proteolytic activity of Paracoccidioides brasiliensis after SDS-PAGE using agarose overlays containing Abz-MKALTLQ-EDDnp

    OpenAIRE

    Puccia, R; M. A. Juliano; L. Juliano; Travassos, L R; Carmona, A. K.

    1999-01-01

    We have characterized, in the Paracoccidioides brasiliensis yeast phase, an exocellular SH-dependent serine proteinase activity against Abz-MKRLTL-EDDnp and analogous fluorescent-quenched peptides, and showed that it is also active against constituents of the basement membrane in vitro. In the present study, we separated the components of P. brasiliensis culture filtrates by electrophoresis and demonstrated that the serine-thiol exocellular proteinase has a diffuse and heterogeneous migration...

  20. Characterization of β-Glucosidase Produced by Aspergillus niger under Solid-State Fermentation and Partially Purified Using MANAE-Agarose

    OpenAIRE

    Anderson Baraldo Junior; Borges, Diogo G.; Tardioli, Paulo W.; Farinas, Cristiane S.

    2014-01-01

    β -Glucosidase (BGL) is a hydrolytic enzyme with specificity for a wide variety of glycoside substrates, being an enzyme with a large range of biotechnological applications. However, enzyme properties can be different depending both on the microorganism and the cultivation procedure employed. Therefore, in order to explore potential biocatalytical applications of novel enzymes, their characterization is essential. In this work, a BGL synthesized by a selected strain of Aspergillus niger culti...

  1. USE OF DNA PURIFIED IN SITU FROM CELLS EMBEDDED IN AGAROSE PLUGS FOR THE MOLECULAR ANALYSIS OF TK-/-MUTANTS RECOVERED IN THE L5178Y TK+/- 3.7.2C MUTAGEN ASSAY SYSTEM

    Science.gov (United States)

    We have reported that tk-/- mutants recovered in the mouse L5178Y TK+/- 3.7.2C mutagen assay have often lost the tk+ allele. llele loss in tk-/- mutants is mented on Southern blots as the absence of a 6.3-kb Nco I fragment seen in both tk+/+ and tk+/- cell DNAs. or the routine sc...

  2. Immobilization of a Commercial Lipase from Penicillium camembertii (Lipase G) by Different Strategies

    OpenAIRE

    Adriano A. Mendes; Larissa Freitas; de Carvalho, Ana Karine F.; de Oliveira, Pedro C.; Heizir F. de Castro

    2011-01-01

    The objective of this work was to select the most suitable procedure to immobilize lipase from Penicillium camembertii (Lipase G). Different techniques and supports were evaluated, including physical adsorption on hydrophobic supports octyl-agarose, poly(hydroxybutyrate) and Amberlite resin XAD-4; ionic adsorption on the anionic exchange resin MANAE-agarose and covalent attachment on glyoxyl-agarose, MANAE-agarose cross-linked with glutaraldehyde, MANAE-agarose-glutaraldehyde, and epoxy-silic...

  3. Fluorographic detection of tritium-labelled proteins in immunoelectropherograms with the water-soluble fluor, sodium salicylate

    International Nuclear Information System (INIS)

    A method is described for detecting by fluorography tritium-labelled proteins in immunoelectropherograms performed on agarose gels. The technique is based on the impregnation of immunoplates with sodium salicylate immediately after electrophoresis without the formation of a mixed agarose-polyacrylamide gel prior to processing the immunoelectropherograms for fluorography. Sodium salicylate, an inexpensive and water-soluble compound, has been found to serve as a satisfactory fluor for enhanced detection of radioactivity in agarose gels. (Auth.)

  4. The Substitute Brain and the Potential of the Gel Model

    OpenAIRE

    Pomfret, Roland; Miranpuri, Gurwattan; Sillay, Karl

    2013-01-01

    This purpose of this paper is to review the recent history of the use of agarose gels. Although originally confined to electrophoresis work, agarose gels have proven themselves useful to a number of disciplines in the modern world, which includes brain infusion studies for research involving the treatment of various neurological conditions, such as Parkinson’s Disease. In reviewing the relevant research leading up to the modern day, this paper attempts to track agarose gels through their stag...

  5. Immobilization of Yarrowia lipolytica Lipase—A Comparison of Stability of Physical Adsorption and Covalent Attachment Techniques

    Science.gov (United States)

    Cunha, Aline G.; Fernández-Lorente, Gloria; Bevilaqua, Juliana V.; Destain, Jacqueline; Paiva, Lúcia M. C.; Freire, Denise M. G.; Fernández-Lafuente, Roberto; Guisán, Jose M.

    Lipase immobilization offers unique advantages in terms of better process control, enhanced stability, predictable decay rates and improved economics. This work evaluated the immobilization of a highly active Yarrowia lipolytica lipase (YLL) by physical adsorption and covalent attachment. The enzyme was adsorbed on octyl-agarose and octadecyl-sepabeads supports by hydrophobic adsorption at low ionic strength and on MANAE-agarose support by ionic adsorption. CNBr-agarose was used as support for the covalent attachment immobilization. Immobilization yields of 71, 90 and 97% were obtained when Y. lipolytica lipase was immobilized into octyl-agarose, octadecyl-sepabeads and MANAE-agarose, respectively. However, the activity retention was lower (34% for octyl-agarose, 50% for octadecyl-sepabeads and 61% for MANAE-agarose), indicating that the immobilized lipase lost activity during immobilization procedures. Furthermore, immobilization by covalent attachment led to complete enzyme inactivation. Thermal deactivation was studied at a temperature range from 25 to 45°C and pH varying from 5.0 to 9.0 and revealed that the hydrophobic adsorption on octadecyl-sepabeads produced an appreciable stabilization of the biocatalyst. The octadecyl-sepabeads biocatalyst was almost tenfold more stable than free lipase, and its thermal deactivation profile was also modified. On the other hand, the Y. lipolytica lipase immobilized on octyl-agarose and MANAE-agarose supports presented low stability, even less than the free enzyme.

  6. DNA Technology in the Classroom.

    Science.gov (United States)

    Williamson, John H.; Campbell, A. Malcolm

    1997-01-01

    Presents a protocol that gives students hands-on experience in generating a meaningful physical map of a circular molecule of DNA. Topics include agarose gel electrophoresis, logic of restriction maps, extracting data from an agarose gel, managing data from gels, experimental protocol, loading gels, electrophoresis, photographing gels, collecting…

  7. Isolation and characterization of porcine mannan-binding proteins of different size and ultrastructure

    DEFF Research Database (Denmark)

    Storgaard, P; Nielsen, EH; Andersen, Ove;

    1996-01-01

    mouse and rat MBP-C (41-45% identity). Both pMBPs exhibited Ca2+-dependent binding to D-mannose immobilized on agarose but no significant binding to N-acetyl-D-glucosamine- or fucose-agarose. The results further suggested the presence of a third pMBP which copurified with pMBP-27 but this protein was...

  8. Hybrid silver nanoparticle/conjugated polyelectrolyte nanocomposites exhibiting controllable metal-enhanced fluorescence

    Science.gov (United States)

    Wang, Xiaoyu; He, Fang; Zhu, Xi; Tang, Fu; Li, Lidong

    2014-03-01

    Metal-enhanced fluorescence of conjugated polyelectrolytes (CPs) is realized using a simple, green hybrid Ag nanocomposite film. Ag nanoparticles (Ag NPs) are pre-prepared by sodium citrate reduction and incorporated into agarose by mixing to form an Ag-containing agarose film (Ag@agarose). Through variation of the amount of Ag NPs in the Ag@agarose film as well as the thickness of the interlayer between CPs and the Ag@agarose film prepared of layer-by-layer assembly of chitosan and sodium alginate, a maximum 8.5-fold increase in the fluorescence of CPs is obtained. After introducing tyrosinase, this system also can be used to detect phenolic compounds with high sensitivity and good visualization under ultraviolet light.

  9. Efficacy of transdermal magnesium ascorbyl phosphate delivery after ultrasound treatment with microbubbles in gel-type surrounding medium in mice.

    Science.gov (United States)

    Liao, Ai-Ho; Lu, Ying-Jui; Hung, Chi-Ray; Yang, Meng-Yu

    2016-04-01

    Liquid microemulsions appropriate for topical application were obtained by increasing their viscosity through the addition of thickening agents. The present study first assessed the usefulness of ultrasound (US) plus US contrast agent, microbubbles (MBs), in agarose gel for enhancing transdermal drug delivery. The effect of US plus MBs in agarose gel on the penetration of the skin by magnesium ascorbyl phosphate (MAP) was explored both in vitro and in vivo. In the in vitro experiments, the stability of MBs was investigated by examining the penetration of MAP by the model drug, Evans blue, in two media: an agarose phantom and pig skin. The penetration depth in the agarose phantom and pig skin increased by 40% and 195%, respectively, when treated with US plus MBs in 0.1% agarose solution combined with MAP (UMB1), and by 48% and 206%, respectively, when treated with US plus MBs in 0.15% agarose solution and MAP (UMB2). The skin-whitening effects in C57BL/6J mice in the UMB1 and UMB2 groups over a 4-week experimental period were significantly increased by 63% and 70%, respectively, in the fourth week. The findings of this study suggest that the survival of MBs with US is affected by the viscosity of the surrounding medium, and that in mice, treatment with US plus MBs in a suitable agarose gel can increase skin permeability and enhance transdermal MAP delivery. PMID:26838887

  10. A New Electrophoresis Technique to Seperate Microsatellite Alleles

    Science.gov (United States)

    Traditional agarose and polyacrylamide gel electrophoresis have been used commonly for microsatellite (simple sequence repeats, SSRs) analysis, but they are labor- intensive and not always able to provide accurate sizes for different alleles. Capillary sequencers provide automated analysis and accur...

  11. A simple method for clonal selection of hepatitis A virus based on recovery of virus from radioimmunofocus overlays

    International Nuclear Information System (INIS)

    Hepatitis A virus (HAV), has been quantitated in cell culture by autoradiographic detection of foci of viral replication developing beneath an agarose overlay following fixation and 'staining' of the cell sheet with radiolabelled antibody (radioimmunofocus assay). Using a modification of this basic technique, a clonal variant of HM-175 strain HAV was isolated from agarose overlying individual radioimmunofoci. Virus recovered from the agarose was amplified in small volume cultures of BS-C-1 cells and identified in supernatant culture fluids by cDNA-RNA hybridizaton. No virus was recovered from agarose which did not overlie a focus of viral replication. This method offers a simple, yet relatively rapid and certain means of selecting clonal variants of non-plaquing viruses such as hepatitis A virus. (Auth.)

  12. Hydrogel-Assisted Transfer of Graphene Oxides into Nonpolar Organic Media for Oil Decontamination.

    Science.gov (United States)

    Cheng, Chongling; Wang, Dayang

    2016-06-01

    In this work, graphene oxide (GO)-loaded agarose hydrogel was transferred into oil such as hexadecane via stepwise solvent exchange with no chemical modification of the GO hydrophilic surface and the agarose network. After transfer, the GOs, loaded in the agarose network, could effectively and efficiently adsorb lipophilic dyes in oil via hydrogen bonding between the polar groups of the GOs and the dyes. The maximum adsorption capacity was 355.9 mg g(-1) for Nile red for instance, which is substantially larger than that of pristine agarose hydrogel and hydrophilic GO powder. The dye concentration for effective adsorption can be as low as 0.5 ppm. Thus, the present work demonstrates the promising potential of using hydrophilic adsorbents for efficient removal of polar impurities from oil. PMID:27112433

  13. Stability in Escherichia coli of an antibiotic resistance plasmid from Bacteroides fragilis.

    OpenAIRE

    Rashtchian, A; Booth, S J

    1981-01-01

    A Bacteroides fragilis strain resistant to penicillin G, tetracycline, and clindamycin was screened for the presence of plasmid deoxyribonucleic acid (DNA). Agarose gel electrophoresis of ethanol-precipitated DNA from cleared lysates of this strain revealed two plasmid DNA bands. The molecular weights of the plasmids were estimated by their relative mobility in agarose gel and compared with standard plasmids with known molecular weights. The molecular weights were 3.40 +/- 0.20 x 10(6) and 1....

  14. Clinical applications of immunofixation: a more sensitive technique for the detection of Bence Jones protein.

    OpenAIRE

    Whicher, J T; Hawkins, L.; Higginson, J.

    1980-01-01

    Immunofixation in agarose gel has been compared with agarose electrophoresis for the detection of Bence Jones protein in urine. The technique has a sensitivity between five and 10 times greater than electrophoresis and allows the identification of multiple Bence Jones proteins and Bence Jones proteins with fast mobility in the presence of other urinary proteins. In four out of 12 patients studied, Bence Jones protein was undetectable by electrophoresis of 300 times concentrated urine but was ...

  15. Atypical M-Protein Localization in Protein Electrophoresis in a Patient With Multiple Myeloma

    OpenAIRE

    Yıldırım, Naciye Demirel; Ayer, Mesut; Hatipoğlu, Esra; Küçükkaya, Reyhan Diz; Yenerel, Mustafa Nuri; Nalçacı, Meliha

    2008-01-01

    Monoclonal gammopathy is a group of B-cell disorders resulting in the secretion of a specific and unique monoclonal immunoglobulin (M-component). The best method for detecting a monoclonal protein is high resolution agarose gel electrophoresis. This test detects abnormalities in the migration of the proteins on electrophoresis and can be performed with samples of serum or urine. An M-protein is usually visible as a localized band on agarose gel electrophoretic peak in the beta, gamma, or rare...

  16. Exfoliative toxin plasmids of bacteriophage group 2 Staphylococcus aureus: sequence homology.

    OpenAIRE

    Warren, R. L.

    1980-01-01

    The plasmid contents of seven exfoliative toxin-producing strains of phage group 2 Staphylococcus aureus were analyzed by agarose gel electrophoresis and deoxyribonucleic acid-deoxyribonucleic acid hybridization. All strains were found to contain a large plasmid with a molecular weight of 27 X 10(6) except for strain RW1005. A comparison of the restriction endonuclease cleavage products by agarose gel electrophoresis showed that the number and size distribution of the fragments of all these T...

  17. Simian virus 40 encapsidation: characterization of early intermediates.

    OpenAIRE

    Milavetz, B; Hopkins, T

    1982-01-01

    Simian virus 40 chromosomes were separated into various species by a two-step purification consisting of low-ionic-strength glycerol gradient sedimentation followed by low-ionic-strength agarose gel electrophoresis. For each species of simian virus 40 chromosome purified, the comigrating DNA and proteins were identified by agarose or polyacrylamide gel electrophoresis, respectively. Two species of chromosomes were identified which contained form I and form II DNA and large amounts of viral pr...

  18. Comparative Analysis of Human and Porcupine (Hystrix Cristata L., 1758) Haemoglobins

    OpenAIRE

    ÇİĞREMİŞ, Yılmaz; ATALAR, Ömer; Kenan ERDOĞAN; Gaffaroğlu, Muhammet; Türköz, Yusuf; YILMAZ, Sadık

    2010-01-01

    Agarose gel electrophoresis was used to determine the electrophoretic pattern of the haemoglobin of Hystrix cristata (H. cristata), and that of a healthy human. Alkaline agarose gel electrophoresis of the haemoglobin of H. cristata revealed that the mobility of the H. cristata haemoglobin was considerably faster than that of human haemoglobin. These comparisons showed obvious differences between the haemoglobin of the two species. 

  19. Electron Beam Sterilization of the Plates with Agaroze Gel Used for Electrophoresis

    Science.gov (United States)

    Ighigeanu, Daniel I.; Martin, Diana I.; Stan, Dana E.; Matei, Constantin I.; Manaila, Elena M.; Craciun, Gabriela D.; Iacob, Nicusor I.; Oproiu, Constantin V.; Ighigeanu, Adelina I.

    2007-04-01

    Electron beam (EB) sterilization applied to the plastic plates with agarose gel used for electrophoresis is presented. The effects of EB irradiation upon the agarose gel and on the process of the proteic fraction separation have been investigated. The investigation were focused on the concentration changes of the six proteic fractions, albumin, alpha 1, alpha 2, beta 1, beta 2 and gamma, versus the dose irradiation as compared with the unirradiated sample.

  20. Complete in vitro DNA replication of SV40 chromatin in digitonin-treated permeable cells.

    OpenAIRE

    Oda,Takuzo; Watanabe,Sekiko; Hanakawa,Shiro; Nakamura, Takashi

    1980-01-01

    A permeable cell system has been developed by treatment with digitonin for studying in vitro DNA replication of chromatin. DNA replication of simian virus 40 nucleoprotein complexes (SV40 chromatin) in digitonin-treated permeable cells was analyzed by electrophoresis in agarose-gel. Autoradiography of the agarose-gel revealed that [32P]dCTP was incorporated in SV40 DNA I, II and replicating intermediates. The time course of the incorporation indicated the complete replication of SV40 DNA and ...

  1. Replication of vertebrate mitochondrial DNA entails transient ribonucleotide incorporation throughout the lagging strand

    OpenAIRE

    Yasukawa, Takehiro; Reyes, Aurelio; Cluett, Tricia J.; Yang, Ming-Yao; Bowmaker, Mark; Jacobs, Howard T.; Holt, Ian J.

    2006-01-01

    Using two-dimensional agarose gel electrophoresis, we show that mitochondrial DNA (mtDNA) replication of birds and mammals frequently entails ribonucleotide incorporation throughout the lagging strand (RITOLS). Based on a combination of two-dimensional agarose gel electrophoretic analysis and mapping of 5′ ends of DNA, initiation of RITOLS replication occurs in the major non-coding region of vertebrate mtDNA and is effectively unidirectional. In some cases, conversion of nascent RNA strands t...

  2. Improved DNA Electrophoresis in Conditions Favoring Polyborates and Lewis Acid Complexation

    OpenAIRE

    Singhal, Hari; Ren, Yunzhao R.; Kern, Scott E.

    2010-01-01

    Spatial compression among the longer DNA fragments occurs during DNA electrophoresis in agarose and non-agarose gels when using certain ions in the conductive buffer, impairing the range of fragment sizes resolved well in a single gel. Substitutions using various polyhydroxyl anions supported the underlying phenomenon as the complexation of Lewis acids to DNA. We saw significant improvements using conditions (lithium borate 10 mM cations, pH 6.5) favoring the formation of borate polyanions an...

  3. Purification of Staphylococcus aureus beta-lactamases by using sequential cation-exchange and affinity chromatography.

    OpenAIRE

    Kernodle, D S; Zygmunt, D J; McGraw, P A; Chipley, J R

    1990-01-01

    Boronic acids are active-site inhibitors of serine beta-lactamases, and a phenylboronic acid-agarose affinity column has been used to purify beta-lactamase from crude cell extracts of several bacterial species. We applied phenylboronic acid-agarose chromatography to the purification of Staphylococcus aureus beta-lactamase. Two factors interfered with the success of the previously described single-step chromatographic protocol. First, staphylococcal beta-lactamase exhibited non-active-site-med...

  4. Polysaccharide-supported planar bilayer lipid model membranes

    OpenAIRE

    Baumgart, T.; Offenhäusser, A.

    2003-01-01

    Bilayer lipid membranes were deposited onto two different thin water swellable polymer cushions predominantly by Langmuir-Blodgett trough methods. Membranes consisting of zwitterionic lipids supported by agarose films were shown to be unstable, as observed by fluorescence microscopy, reflection interference contrast microscopy, and the impossibility of bilayer spreading (Radler, J.; Strey, H.; Sackmann, E. Langmuir 1995, 11, 4539-4548) on the agarose surface. Chitosan, formerly observed to pe...

  5. Immobilization of a Commercial Lipase from Penicillium camembertii (Lipase G by Different Strategies

    Directory of Open Access Journals (Sweden)

    Adriano A. Mendes

    2011-01-01

    Full Text Available The objective of this work was to select the most suitable procedure to immobilize lipase from Penicillium camembertii (Lipase G. Different techniques and supports were evaluated, including physical adsorption on hydrophobic supports octyl-agarose, poly(hydroxybutyrate and Amberlite resin XAD-4; ionic adsorption on the anionic exchange resin MANAE-agarose and covalent attachment on glyoxyl-agarose, MANAE-agarose cross-linked with glutaraldehyde, MANAE-agarose-glutaraldehyde, and epoxy-silica-polyvinyl alcohol composite. Among the tested protocols, the highest hydrolytic activity (128.2 ± 8.10 IU·g−1 of support was achieved when the lipase was immobilized on epoxy-SiO2-PVA using hexane as coupling medium. Lipase immobilized by ionic adsorption on MANAE-agarose also gave satisfactory result, attaining 55.6 ± 2.60 IU·g−1 of support. In this procedure, the maximum loading of immobilized enzyme was 9.3 mg·g−1 of gel, and the highest activity (68.8 ± 2.70 IU·g−1 of support was obtained when 20 mg of protein·g−1 was offered. Immobilization carried out in aqueous medium by physical adsorption on hydrophobic supports and covalent attachment on MANAE-agarose-glutaraldehyde and glyoxyl-agarose was shown to be unfeasible for Lipase G. Thermal stability tests revealed that the immobilized derivative on epoxy-SiO2-PVA composite using hexane as coupling medium had a slight higher thermal stability than the free lipase.

  6. Immobilization of a Commercial Lipase from Penicillium camembertii (Lipase G) by Different Strategies.

    Science.gov (United States)

    Mendes, Adriano A; Freitas, Larissa; de Carvalho, Ana Karine F; de Oliveira, Pedro C; de Castro, Heizir F

    2011-01-01

    The objective of this work was to select the most suitable procedure to immobilize lipase from Penicillium camembertii (Lipase G). Different techniques and supports were evaluated, including physical adsorption on hydrophobic supports octyl-agarose, poly(hydroxybutyrate) and Amberlite resin XAD-4; ionic adsorption on the anionic exchange resin MANAE-agarose and covalent attachment on glyoxyl-agarose, MANAE-agarose cross-linked with glutaraldehyde, MANAE-agarose-glutaraldehyde, and epoxy-silica-polyvinyl alcohol composite. Among the tested protocols, the highest hydrolytic activity (128.2 ± 8.10 IU·g(-1) of support) was achieved when the lipase was immobilized on epoxy-SiO(2)-PVA using hexane as coupling medium. Lipase immobilized by ionic adsorption on MANAE-agarose also gave satisfactory result, attaining 55.6 ± 2.60 IU·g(-1) of support. In this procedure, the maximum loading of immobilized enzyme was 9.3 mg·g(-1) of gel, and the highest activity (68.8 ± 2.70 IU·g(-1) of support) was obtained when 20 mg of protein·g(-1) was offered. Immobilization carried out in aqueous medium by physical adsorption on hydrophobic supports and covalent attachment on MANAE-agarose-glutaraldehyde and glyoxyl-agarose was shown to be unfeasible for Lipase G. Thermal stability tests revealed that the immobilized derivative on epoxy-SiO(2)-PVA composite using hexane as coupling medium had a slight higher thermal stability than the free lipase. PMID:21811674

  7. Electrophoretic mobility of PM2 DNA treated with ultimate chemical carcinogens or with ultraviolet light

    International Nuclear Information System (INIS)

    Superhelical DNA of the Pseudomonas phage PM2 was irradiated with UV-light or reacted with covalently binding carcinogens, such as 7-bromomethyl-benz[a]anthracene, (Ac)2ONFln, K-region epoxides, and alkylating agents. Migration velocity of the DNA products was determined using agarose gel electrophoresis. In gels of more than 1.3%-1.9% agarose, modified PM2 DNA exhibited a dose-(concentration-)dependent decrease of migration velocity. This phenomenon is probably due to a decrease in superhelix density which caused the compact DNA coil to assume eventually an open-circular conformation. Comparison of the extent of DNA modification with the decrease of migration velocity revealed that the superhelical structure sensitively reflected the chemical DNA alterations. DNA species exhibiting in 1.6% agarose gels, a migration velocity of up to 30% of that of control DNA showed an increase of velocity in 0.4% agarose. Therefore, in 1.3%-1.9% agarose gels, the decrease of superhelix density is accompanied by an increase of the frictional coefficient, whereas in 0.4%-0.9% agarose gels the same decrease of superhelix density apparently led to a higher degree of flexibility of the macromolecule and/or exposure of additional electric charges. (orig.)

  8. Molecular morphology of cyanobacterial phycobilisomes

    Energy Technology Data Exchange (ETDEWEB)

    Siegelman, H.W.; Kycia, J.H.

    1982-09-01

    Phycobilisomes were isolated from several cyanobacteria following cell lysis with Triton X-100. They were purified by phosphate precipitation and hydrophobic-interaction chromatography. Their phycobiliprotein compositions were quantitatively determined by application of sets of simultaneous absorbance equations to gel chromatographic separations of the chromoproteins. Phycobilisomes purified from several cyanobacteria had characteristic elution times on agarose gel chromatography. Combining electron microscope observations of phycobilisome structure, phycobiliprotein composition, and agarose gel chromatography estimates of molecular weight permitted the calculation of many details of phycobilisome molecular structure. Complementary chromatic adaptation resulted in a change of phycobilisome composition and structure. The polypeptide compositions of phycobilisomes were examined by sodium dodecyl sulfate-agarose gel chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The phycobilisomes were composed of phycobilipeptides derived from the constituent phycobiliproteins. Higher molecular-weight phycobilipeptide aggregates were also observed. The dominant forces responsible for the maintenance of phycobilisome structure are concluded to be hydropohobic interactions.

  9. Rheological Characterization of Ethanolamine Gel Propellants

    Science.gov (United States)

    V. S Jyoti, Botchu; Baek, Seung Wook

    2016-07-01

    Ethanolamine is considered to be an environmentally friendly propellant system because it has low toxicity and is noncarcinogenic in nature. In this article, efforts are made to formulate and prepare ethanolamine gel systems, using pure agarose and hybrids of paired gelling agents (agarose + polyvinylpyrrolidine (PVP), agarose + SiO2, and PVP + SiO2), that exhibit a measurable yield stress, thixotropic behavior under shear rate ranges of 1-1,000 s-1 and a viscoelastic nature. To achieve these goals, multiple rheological experiments (including flow and dynamic studies) are performed. In this article, results are presented from experiments measuring the apparent viscosity, yield stress, thixotropy, dynamic strain, frequency sweep, and tan δ behaviors, as well as the effects of the test temperature, in the gel systems. The results show that the formulated ethanolamine gels are thixotropic in nature with yield stress between 30 and 60 Pa. The apparent viscosity of the gel decreases as the test temperature increases, and the apparent activation energy is the lowest for the ethanolamine-(PVP + SiO2) gel system. The dynamic rheology study shows that the type of gellant, choice of hybrid gelling materials and their concentration, applied frequencies, and strain all vitally affect the viscoelastic properties of the ethanolamine gel systems. In the frequency sweep experiment, the ethanolamine gels to which agarose, agarose + PVP, and agarose + SiO2 were added behave like linear frequency-dependent viscoelastic liquids, whereas the ethanolamine gel to which PVP + SiO2 was added behaves like a nearly frequency-independent viscoelastic solid. The variation in the tan δ of these gelled propellants as a function of frequency is also discussed.

  10. Drug release into hydrogel-based subcutaneous surrogates studied by UV imaging

    DEFF Research Database (Denmark)

    Ye, Fengbin; Larsen, Susan Weng; Yaghmur, Anan; Jensen, Henrik; Larsen, Claus Selch; Ostergaard, Jesper

    2012-01-01

    triglyceride (MCT) into 0.5% (w/v) agarose or 25% (w/v) F127-based hydrogels was investigated by monitoring the concentration profiles of the drug in the gels. The effect of pH on piroxicam distribution and diffusion coefficients was studied. For both hydrogel systems, the diffusion of piroxicam in the gels...... of piroxicam upon the injection of aqueous or MCT solutions into an agarose-based hydrogel were investigated by UV imaging. The spatial distribution of piroxicam around the injection site in the gel matrix was monitored in real-time. The disappearance profiles of piroxicam from the injected aqueous...

  11. Characterization of plasmids in Erwinia stewartii.

    OpenAIRE

    Coplin, D. L.; Rowan, R G; Chisholm, D A; Whitmoyer, R E

    1981-01-01

    Plasmids in 39 strains of Erwinia stewartii were examined by agarose gel electrophoresis. Most virulent strains had from 11 to 13 plasmids ranging in molecular mass from 2.8 to 210 megadaltons and contained plasmids of 210, 70, 49, 43, 29.5, 16.8, 8.8, and 2.8 megadaltons. Plasmids in strains SW2 and SS104 were characterized by both electron microscopy and agarose gel electrophoresis and may be useful as convenient references for sizing plasmids by electrophoresis. Specific size classes of pl...

  12. Visualization of DNA damage in individual cells

    International Nuclear Information System (INIS)

    A simple technique of micro-agarose gel electrophoresis has been developed to permit an evaluation of DNA damage in individual cells. Cells are embeded in agarose gel on microscope slides, lysed by detergents and then electrophoresed for a short time. In damaged cells, DNA migrated from the nuclei toward the anode, displaying 'comets' visualized by staining with a DNA-specific fluorochrome, acridine orange. The technique was applicable to quantifying DNA damage in individual cells exposed to Gy level of reactor radiation. (author)

  13. Effects of 5-azacytidine on expression of endogenous retrovirus-related sequences in C3H 10T1/2 cells.

    OpenAIRE

    Hsiao, W L; Gattoni-Celli, S; Weinstein, I B

    1986-01-01

    In a previous study (22) we found that transient exposure of C3H 10T1/2 mouse embryo fibroblasts to 5-azacytidine (5-azaC) induced several changes in growth properties. The treated cells showed progressive changes in morphology, saturation density, growth rate, and serum dependence. By passage 5, the cells had acquired the ability to grow in 0.3% agarose, and by passage 30, they had given rise to fully transformed foci that grew in agarose, agar, and liquid suspension. This progression was ra...

  14. Effects of 5-azacytidine on the progressive nature of cell transformation.

    OpenAIRE

    Hsiao, W L; Gattoni-Celli, S; Weinstein, I B

    1985-01-01

    C3H 10T1/2 mouse embryo fibroblasts were exposed to 3 microM 5-azacytidine for 24 h and then serially passaged in the absence of 5-azacytidine and examined for subsequent changes in growth properties. The treated cells showed changes in morphology, saturation density, growth rate, and serum dependence. By the 5th passage they acquired the ability to grow in 0.3% agarose, and by the 30th passage they gave rise to fully transformed foci that grew in agarose, in agar, and in liquid suspension. T...

  15. Drug: D01032 [KEGG MEDICUS

    Lifescience Database Archive (English)

    Full Text Available D01032 Crude, Drug Agar (JP16/NF); Powdered agar (JP16); Agar (TN) Agarose [CPD:C01...100 Gelidiaceae Gelidium amansii mucous (freeze dry) Major component: Agarose [CPD:C01399] Therapeutic category of dr...ugs in Japan [BR:br08301] 5 Crude drugs and Chinese medicine formulations 51 Crude dr...ugs 510 Crude drugs 5100 Crude drugs D01032 Agar (JP16/NF); Powdered agar (JP16) Crude drugs [BR:br08305] Algae Red algae D01032 Agar PubChem: 7848095 ...

  16. Improving the Thermostability and Optimal Temperature of a Lipase from the Hyperthermophilic Archaeon Pyrococcus furiosus by Covalent Immobilization

    Directory of Open Access Journals (Sweden)

    Roberta V. Branco

    2015-01-01

    Full Text Available A recombinant thermostable lipase (Pf2001Δ60 from the hyperthermophilic Archaeon Pyrococcus furiosus (PFUL was immobilized by hydrophobic interaction on octyl-agarose (octyl PFUL and by covalent bond on aldehyde activated-agarose in the presence of DTT at pH = 7.0 (one-point covalent attachment (glyoxyl-DTT PFUL and on glyoxyl-agarose at pH 10.2 (multipoint covalent attachment (glyoxyl PFUL. The enzyme’s properties, such as optimal temperature and pH, thermostability, and selectivity, were improved by covalent immobilization. The highest enzyme stability at 70°C for 48 h incubation was achieved for glyoxyl PFUL (around 82% of residual activity, whereas glyoxyl-DTT PFUL maintained around 69% activity, followed by octyl PFUL (27% remaining activity. Immobilization on glyoxyl-agarose improved the optimal temperature to 90°C, while the optimal temperature of octyl PFUL was 70°C. Also, very significant changes in activity with different substrates were found. In general, the covalent bond derivatives were more active than octyl PFUL. The E value also depended substantially on the derivative and the conditions used. It was observed that the reaction of glyoxyl-DTT PFUL using methyl mandelate as a substrate at pH 7 presented the best results for enantioselectivity E=22 and enantiomeric excess (ee (% = 91.

  17. Three-dimensional quantification of susceptibility artifacts from various metals in magnetic resonance images.

    Science.gov (United States)

    Imai, Haruki; Tanaka, Yoji; Nomura, Naoyuki; Tsutsumi, Yusuke; Doi, Hisashi; Kanno, Zuisei; Ohno, Kikuo; Ono, Takashi; Hanawa, Takao

    2013-09-01

    Susceptibility artifacts generated in magnetic resonance (MR) images were quantitatively evaluated for various metals using a three-dimensional (3-D) artifact rendering to demonstrate the correlation between magnetic susceptibility and artifact volume. Ten metals (stainless steel, Co-Cr alloy, Nb, Ti, Zr, Mo, Al, Sn, Cu and Ag) were prepared, and their magnetic susceptibilities measured using a magnetic balance. Each metal was embedded in a Ni-doped agarose gel phantom and the MR images of the metal-containing phantoms were taken using 1.5 and 3.0 T MR scanners under both fast spin echo and gradient echo conditions. 3-D renderings of the artifacts were constructed from the images and the artifact volumes were calculated for each metal. The artifact volumes of metals decreased with decreasing magnetic susceptibility, with the exception of Ag. Although Sn possesses the lowest absolute magnetic susceptibility (1.8×10(-6)), the artifact volume from Cu (-7.8×10(-6)) was smaller than that of Sn. This is because the magnetic susceptibility of Cu was close to that of the agarose gel phantom (-7.3×10(-6)). Since the difference in magnetic susceptibility between the agarose and Sn is close to that between the agarose and Ag (-17.5×10(-6)), their artifact volumes were almost the same, although they formed artifacts that were reversed in all three dimensions. PMID:23707948

  18. Detection of Listeria monocytogenes by using the polymerase chain reaction

    International Nuclear Information System (INIS)

    A method was developed for detection of Listeria monocytogens by polymerase chain reaction amplification followed by agarose gel electrophoresis or dot blot analysis with 32P-labeled internal probe. The technique identified 95 of 95 L. monocytogenes strains, 0 of 12 Listeria strains of other species, and 0 of 12 non-Listeria strains

  19. An investigation of the HUMVWA31A locus in British Caucasians

    DEFF Research Database (Denmark)

    Drozd, M A; Archard, L; Lincoln, P J;

    1994-01-01

    A number of short tandem repeat (STR) loci are currently being examined for their usefulness as markers of identity; HUMVWA31A is one such locus. We used a high-sieving agarose technique to type 200 British Caucasians for this locus. Comparison of the resultant allele frequencies with other...

  20. A novel inert crystal delivery medium for serial femtosecond crystallography.

    Science.gov (United States)

    Conrad, Chelsie E; Basu, Shibom; James, Daniel; Wang, Dingjie; Schaffer, Alexander; Roy-Chowdhury, Shatabdi; Zatsepin, Nadia A; Aquila, Andrew; Coe, Jesse; Gati, Cornelius; Hunter, Mark S; Koglin, Jason E; Kupitz, Christopher; Nelson, Garrett; Subramanian, Ganesh; White, Thomas A; Zhao, Yun; Zook, James; Boutet, Sébastien; Cherezov, Vadim; Spence, John C H; Fromme, Raimund; Weierstall, Uwe; Fromme, Petra

    2015-07-01

    Serial femtosecond crystallography (SFX) has opened a new era in crystallo-graphy by permitting nearly damage-free, room-temperature structure determination of challenging proteins such as membrane proteins. In SFX, femtosecond X-ray free-electron laser pulses produce diffraction snapshots from nanocrystals and microcrystals delivered in a liquid jet, which leads to high protein consumption. A slow-moving stream of agarose has been developed as a new crystal delivery medium for SFX. It has low background scattering, is compatible with both soluble and membrane proteins, and can deliver the protein crystals at a wide range of temperatures down to 4°C. Using this crystal-laden agarose stream, the structure of a multi-subunit complex, phycocyanin, was solved to 2.5 Å resolution using 300 µg of microcrystals embedded into the agarose medium post-crystallization. The agarose delivery method reduces protein consumption by at least 100-fold and has the potential to be used for a diverse population of proteins, including membrane protein complexes. PMID:26177184

  1. MRI thermometry in phantoms by use of the proton resonance frequency shift method: application to interstitial laser thermotherapy

    Energy Technology Data Exchange (ETDEWEB)

    Olsrud, Johan; Wirestam, Ronnie; Brockstedt, Sara; Persson, Bertil R.R. [Department of Radiation Physics, Lund University Hospital, SE-221 85 Lund (Sweden); Nilsson, Annika M.K. [Department of Physics, Lund Institute of Technology, SE-221 00 Lund (Sweden); Tranberg, Karl-Goeran [Department of Surgery, Lund University Hospital, SE-221 85 Lund (Sweden); Staahlberg, Freddy [Department of Radiation Physics, Lund University Hospital, SE-221 85 Lund (Sweden); Department of Diagnostic Radiology, Lund University Hospital, SE-221 85 Lund (Sweden)

    1998-09-01

    In this work the temperature dependence of the proton resonance frequency was assessed in agarose gel with a high melting temperature (95 deg. C) and in porcine liver in vitro at temperatures relevant to thermotherapy (25-80 deg. C). Furthermore, an optically tissue-like agarose gel phantom was developed and evaluated for use in MRI. The phantom was used to visualize temperature distributions from a diffusing laser fibre by means of the proton resonance frequency shift method. An approximately linear relationship (0.0085 ppm deg. C{sup -1}) between proton resonance frequency shift and temperature change was found for agarose gel, whereas deviations from a linear relationship were observed for porcine liver. The optically tissue-like agarose gel allowed reliable MRI temperature monitoring, and the MR relaxation times (T{sub 1} and T{sub 2}) and the optical properties were found to be independently alterable. Temperature distributions around a diffusing laser fibre, during irradiation and subsequent cooling, were assessed with high spatial resolution (voxel size = 4.3 mm{sup 3}) and with random uncertainties ranging from 0.3 deg. C to 1.4 deg. C (1 SD) with a 40 s scan time. (author)

  2. Visualising DNA in Classrooms Using Nile Blue

    Science.gov (United States)

    Milne, Christine; Roche, Scott; McKay, David

    2008-01-01

    Giving students the opportunity to extract, manipulate and visualise DNA molecules enhances a constructivist approach to learning about modern techniques in biology and biotechnology Visualisation usually requires agarose gel electrophoresis and staining. In this article, we report on an alternative DNA stain, Nile Blue A, that may be used in the…

  3. Zn2+ blocks annealing of complementary single-stranded DNA in a sequence-selective manner

    Science.gov (United States)

    A simple low-temperature EDTA-free agarose gel electrophoresis procedure (LTEAGE) coupled with UV-Vis spectrum and fluorescence quenching analyses was developed and the Zn2+-single-stranded (ss) DNA interaction was investigated under near-physiological conditions. It was found that Zn2+ blocked the...

  4. A new strategy for mapping the human genome.

    OpenAIRE

    Shaw, D. J.

    1986-01-01

    Recent advances in agarose gel electrophoresis of large DNA fragments raise the possibility of an entirely new approach to mapping mammalian genomes. In this article is discussed the potential of this technology for tackling problems such as construction of linkage maps, identifying chromosome translocation breakpoints, and moving from linked markers to genes causing diseases such as the muscular dystrophies and Huntington's chorea.

  5. The repton model of gel electrophoresis

    OpenAIRE

    Barkema, G. T.; Newman, M. E. J.

    1996-01-01

    We discuss the repton model of agarose gel electrophoresis of DNA. We review previous results, both analytic and numerical, as well as presenting a new numerical algorithm for the efficient simulation of the model, and suggesting a new approach to the model's analytic solution.

  6. Gel Electrophoresis--The Easy Way for Students

    Science.gov (United States)

    VanRooy, Wilhelmina; Sultana, Khalida

    2010-01-01

    This article describes a simple, inexpensive, easy to conduct gel-electrophoresis activity using food dyes. It is an alternative to the more expensive counterparts which require agarose gel, DNA samples, purchased chamber and Tris-borate-EDTA buffer. We suggest some learning activities for senior biology students along with comments on several…

  7. Malignant transformation of guinea pig cells after exposure to ultraviolet-irradiated guinea pig cytomegalovirus

    International Nuclear Information System (INIS)

    Guinea pig cells were malignantly transformed in vitro by ultraviolet (uv)-irradiated guinea pig cytomegalovirus (GPCMV). When guinea pig hepatocyte monolayers were infected with uv-irradiated GPCMV, three continuous epithelioid cell lines which grew in soft agarose were established. Two independently derived GPCMV-transformed liver cells and a cell line derived from a soft agarose clone of one of these lines induced invasive tumors when inoculated subcutaneously or intraperitoneally into nude mice. The tumors were sarcomas possibly derived from hepatic stroma or sinusoid. Transformed cell lines were also established after infection of guinea pig hepatocyte monolayers with human cytomegalovirus (HCMV) or simian virus 40 (SV40). These cell lines also formed colonies in soft agarose and induced sarcomas in nude mice. It is concluded that (i) GPCMV can malignantly transform guinea pig cells; (ii) cloning of GPCMV-transformed cells in soft agarose produced cells that induced tumors with a shorter latency period but with no alteration in growth rate or final tumor size; and (iii) the tumors produced by GPCMV-and HCMV-transformed guinea pig cells were more similar to each other in growth rate than to those induced by SV40-transformed guinea pig cells

  8. How-To-Do-It: Recombinant DNA Technology in the High School Biology Laboratory.

    Science.gov (United States)

    Myers, Richard

    1988-01-01

    Describes a basic biotechnology investigation that includes restriction and ligation of plasmid DNA, transformation of bacteria and cloning of these bacterial cells. Discusses laboratory procedures and another activity in the identification of unknown plasmids by studying agarose gel electrophoresis photographs. (CW)

  9. Use PCR and a Single Hair To Produce a "DNA Fingerprint."

    Science.gov (United States)

    Campbell, A. Malcolm; And Others

    1997-01-01

    Presents a laboratory procedure that involves students extracting their own DNA from a single hair follicle, using the polymerase chain reaction (PCR) to amplify a polymorphic locus, performing electrophoresis on the PCR products on an agarose gel, and visualizing the alleles to generate a "DNA fingerprint." Discusses theoretical background,…

  10. Effect of cold storage on collagen-based hydrogels for the three-dimensional culture of adipose-derived stem cells

    International Nuclear Information System (INIS)

    Collagen gels have been extensively used as three-dimensional (3D) cell culture systems. To enhance their mechanical properties, the manufacture of collagen-based gels with agarose has been proposed. However, little is known about the stability of these gels under cold storage conditions. The consequences of cold storage on biological tissues for clinical applications are known to be significant; yet, they have not been considered on hydrogels used for in vitro experiments. This work studies the effect of extended cold storage on the stability of collagen and collagen-agarose hydrogels using rheometry and scanning electron microscopy. In addition, cell-matrix interactions of adipose-derived stem cells (ADSC) have been studied using these gels. Results show that both the storage modulus (G′) and loss modulus (G″) of pure collagen gels gradually decrease with extended cold storage along the 30 days of the study, while G′ and G″ increase in collagen-agarose gels under the same conditions. Moreover, significant changes in both moduli of collagen-agarose gels were only found after 30 days of cold storage, while in the case of collagen gels significant changes were already detected after 7 days. Finally, a reduction in the ability of ADSC to remodel the gel after prolonged cold storage was observed. To the best of our knowledge, this is the first work proving that cold storage of hydrogels prior to cell culture might have a significant impact on their mechanical properties and cell–matrix interactions. (paper)

  11. Using Restriction Mapping to Teach Basic Skills in the Molecular Biology Lab

    Science.gov (United States)

    Walsh, Lauren; Shaker, Elizabeth; De Stasio, Elizabeth A.

    2007-01-01

    Digestion of DNA with restriction enzymes, calculation of volumes and concentrations of reagents for reactions, and the separation of DNA fragments by agarose gel electrophoresis are common molecular biology techniques that are best taught through repetition. The following open-ended, investigative laboratory exercise in plasmid restriction…

  12. A closed-tube assay for genotyping of the 32-bp deletion polymorphism in the chemokine receptor 5 (CCR5) gene

    DEFF Research Database (Denmark)

    Rasmussen, Henrik Berg; Werge, Thomas

    2007-01-01

    the presence of Sybr Green I for allele discrimination. After having established robust conditions for the assay, we used it to genotype 590 unknown DNA samples. A blinded comparison with a procedure based upon agarose gel electrophoresis of amplified material revealed complete concordance between the...

  13. Use of polymerase chain reaction for detection of Chlamydia trachomatis

    DEFF Research Database (Denmark)

    Østergaard, Lars; Birkelund, Svend; Christiansen, Gunna

    1990-01-01

    A polymerase chain reaction (PCR) assay was developed for detection of Chlamydia trachomatis DNA. From the published sequence of the common C. trachomatis plasmid, two primer sets were selected. Detection of amplified sequences was done by agarose gel electrophoresis of cleaved or uncleaved...

  14. Interaction of the Chlamydia trachomatis histone H1-like protein (Hc1) with DNA and RNA causes repression of transcription and translation in vitro

    DEFF Research Database (Denmark)

    Pedersen, Lotte Bang; Birkelund, S; Christiansen, G

    1994-01-01

    concentrations. These results were found to coincide with the formation of condensed Hc1-DNA and Hc1-RNA complexes as revealed by agarose gel electrophoresis and electron microscopy. The implications of these results for possible functions of Hc1 in vivo are discussed. Udgivelsesdato: 1994-Mar...

  15. Detection of foodborne pathogens using microarray technology

    Science.gov (United States)

    Assays based on the polymerase chain reaction (PCR) are now accepted methods for rapidly confirming the presence or absence of specific pathogens in foods and other types of samples. Conventional PCR requires the use of agarose gel electrophoresis to detect the PCR product; whereas, real-time PCR c...

  16. Compensation Of Smile Effect Distortion In Electrophoretic Gel Image

    OpenAIRE

    Dvořáček, T.

    2015-01-01

    This paper is engaged in the issue of automatic detection and removal of smile effect geometrical distortion in agarose gel electrophoresis images. Based on created databank of electrophoretic phantoms, an algorithm that is able to repair mentioned smile effect distortion was created. In this paper, two gel images with applied removal algorithm are shown with percentage description of reparation level.

  17. Problem-Solving Test: Southwestern Blotting

    Science.gov (United States)

    Szeberényi, József

    2014-01-01

    Terms to be familiar with before you start to solve the test: Southern blotting, Western blotting, restriction endonucleases, agarose gel electrophoresis, nitrocellulose filter, molecular hybridization, polyacrylamide gel electrophoresis, proto-oncogene, c-abl, Src-homology domains, tyrosine protein kinase, nuclear localization signal, cDNA,…

  18. Understanding Electrophoresis through the Investigation of Size, Shape, and Charge of pH Indicators

    Science.gov (United States)

    Brenner, Ryan K.; Hess, Kenneth R.; Morford, Jennifer L.

    2015-01-01

    A laboratory experiment was designed for upper-level students in a Chemical Analysis course to illustrate the theoretical and practical applications of 0.8% agarose gel electrophoresis and to reinforce an understanding of weak acids/bases using easy-to-visualize pH indicators. The careful choice of indicators included acid and base types with…

  19. Haemoglobin electrophoresis in diagnosing a case of sickle cell anaemia associated with β-thalassemia

    OpenAIRE

    Geraldine, M; Justin, V.; Sheila, U; Venkatesh, T.

    2001-01-01

    Alkaline haemoglobin electrophoresis is a useful tool in diagnosing β-thalassemia and sickle-cell anaemia. In this report, using this simple technique, β-thalassemia associated with sickle-cell anaemia is diagnosed. This is the first case we have diagnosed in our laboratory using agarose gel electrophoresis.

  20. Genetic and antigenic characterization of Borrelia coriaceae, putative agent of epizootic bovine abortion.

    OpenAIRE

    LeFebvre, R B; Perng, G C

    1989-01-01

    Borrelia coriaceae was characterized genetically and antigenically by utilizing the following techniques: restriction endonuclease analysis, Southern blotting and genomic hybridization, pulsed-field electrophoresis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and immunoblotting. The B. coriaceae genome revealed unique and characteristic banding patterns both by agarose gel electrophoresis and by hybridization when compared with several Borrelia burgdorferi isolates. Pulsed-fiel...

  1. A plasmid in Legionella pneumophila.

    OpenAIRE

    Knudson, G B; Mikesell, P

    1980-01-01

    Sixteen strains from the six serogroups of Legionella pneumophila were examined for the presence of extrachromosomal genetic elements by a modified cleared lysate procedure, dye-buoyant centrifugation, and agarose gel electrophoresis. Two strains, Atlanta-1 and Atlanta-2 from serogroup II, each contained a plasmid of cryptic function with a molecular weight of ca. 30 megadaltons.

  2. Analysis of the RNA Content of the Yeast "Saccharomyces Cerevisiae"

    Science.gov (United States)

    Deutch, Charles E.; Marshall, Pamela A.

    2008-01-01

    In this article, the authors describe an interconnected set of relatively simple laboratory experiments in which students determine the RNA content of yeast cells and use agarose gel electrophoresis to separate and analyze the major species of cellular RNA. This set of experiments focuses on RNAs from the yeast "Saccharomyces cerevisiae", a…

  3. Cryptic plasmids in Lactobacillus strains isolated from the murine gastrointestinal tract.

    OpenAIRE

    Lin, J H; Savage, D C

    1985-01-01

    Ten of twenty Lactobacillus strains isolated from the gastrointestinal tracts of animals of several species contained plasmids of 80 to 90 megadaltons or less than 2.6 megadaltons in size, as analyzed by agarose gel electrophoresis. The large plasmids were found only in strains originally isolated from the keratinized epithelium of the murine stomach.

  4. Modeling the Dynamics of Gel Electrophorresis in the High School Classroom

    Science.gov (United States)

    Saucedo, Skyler R.

    2013-01-01

    Gel electrophoresis, used by geneticists and forensic experts alike, is an immensely popular technique that utilizes an electric field to separate molecules and proteins by size and charge. At the microscopic level, a dye or complex protein like DNA is passed through agarose, a gelatinous three-dimensional matrix of pores and nano-sized tunnels.…

  5. Affinity chromatography of serine proteases on the triazine dye ligand Cibacron Blue F3G-A

    DEFF Research Database (Denmark)

    Koch, C; Borg, L; Skjødt, K;

    1998-01-01

    The interaction between complement component factor B and the triazine dye ligand Cibacron Blue F3G-A coupled to a cross-linked agarose matrix (Blue Sepharose) was found to involve the Bb part of the molecule, and to be inhibited by benzamidine. Human, chicken and rainbow trout factor B which had...

  6. Recovery, Purification, and Cloning of High-Molecular-Weight DNA from Soil Microorganisms▿

    OpenAIRE

    Mark R Liles; Williamson, Lynn L.; Rodbumrer, Jitsupang; Torsvik, Vigdis; Goodman, Robert M.; Handelsman, Jo

    2008-01-01

    We describe here an improved method for isolating, purifying, and cloning DNA from diverse soil microbiota. Soil microorganisms were extracted from soils and embedded and lysed within an agarose plug. Nucleases that copurified with the metagenomic DNA were removed by incubating plugs with a high-salt and -formamide solution. This method was used to construct large-insert soil metagenomic libraries.

  7. A novel inert crystal delivery medium for serial femtosecond crystallography

    Directory of Open Access Journals (Sweden)

    Chelsie E. Conrad

    2015-07-01

    Full Text Available Serial femtosecond crystallography (SFX has opened a new era in crystallography by permitting nearly damage-free, room-temperature structure determination of challenging proteins such as membrane proteins. In SFX, femtosecond X-ray free-electron laser pulses produce diffraction snapshots from nanocrystals and microcrystals delivered in a liquid jet, which leads to high protein consumption. A slow-moving stream of agarose has been developed as a new crystal delivery medium for SFX. It has low background scattering, is compatible with both soluble and membrane proteins, and can deliver the protein crystals at a wide range of temperatures down to 4°C. Using this crystal-laden agarose stream, the structure of a multi-subunit complex, phycocyanin, was solved to 2.5 Å resolution using 300 µg of microcrystals embedded into the agarose medium post-crystallization. The agarose delivery method reduces protein consumption by at least 100-fold and has the potential to be used for a diverse population of proteins, including membrane protein complexes.

  8. Polymers in cell encapsulation from an enveloped cell perspective

    NARCIS (Netherlands)

    de Vos, Paul; Lazarjani, Hamideh Aghajani; Poncelet, Denis; Faas, Marijke M.

    2014-01-01

    In the past two decades, many polymers have been proposed for producing immunoprotective capsules. Examples include the natural polymers alginate, agarose, chitosan, cellulose, collagen, and xanthan and synthetic polymers poly(ethylene glycol), polyvinyl alcohol, polyurethane, poly(ether-sulfone), p

  9. Cartilage-derived extracellular matrix extract promotes chondrocytic phenotype in three-dimensional tissue culture.

    Science.gov (United States)

    Youngstrom, Daniel W; Cakstina, Inese; Jakobsons, Eriks

    2016-05-01

    Cell transplantation is a promising regenerative therapy for cartilage degeneration. However, obtaining sufficient numbers of cells for this purpose is a challenge, due a lack of autologous donor tissue and the difficulty of culturing chondrocytes in vitro. Tissue engineering strategies that induce or maintain chondrocytic phenotype may solve these problems by (1) broadening the range of available donor tissue, and (2) facilitating the expansion of these cells while controlling phenotypic drift. In this study, bone marrow-derived mesenchymal stem cells (MSCs) and cartilage-derived cells (CDCs) were cultured on composite hydrogels containing agarose and homogenized cartilage extracellular matrix (ECM). MSCs cultured on agarose-ECM scaffolds did not show significant signs of chondrogenic differentiation in the absence of additional cues. However, CDCs cultured on agarose-ECM scaffolds proliferated more rapidly than their ECM-free counterparts and MSCs, while retaining chondrocytic morphology. These results were corroborated via expression of cartilage marker genes: in autologous constructs, SOX 9 expression was upregulated by 12.6 ± 5.3-fold, and COL II was upregulated by 2.0 ± 0.3-fold. Agarose-ECM composite hydrogels are therefore useful for expanding partially differentiated CDCs for applications in regenerative medicine. PMID:25707441

  10. EFFECTIVE METHOD TO EXTRACT DNA FROM ENVIRONMENTAL SAMPLES FOR POLYMERASE CHAIN REACTION AMPLIFICATION AND DNA FINGERPRINT ANALYSIS

    Science.gov (United States)

    A rapid direct-extraction method was used to obtain DNA from environmental soil samples. eat, enzymes, and guanidine isothiocyanate were utilized to lyse cells. he DNA was purified by agarose gel electrophoresis, amplified with 16S based primers by use of the polymerase chain rea...

  11. Molecular Cloning and Analysis of a DNA Repetitive Element from the Mouse Genome

    Science.gov (United States)

    Geisinger, Adriana; Cossio, Gabriela; Wettstein, Rodolfo

    2006-01-01

    We report the development of a 3-week laboratory activity for an undergraduate molecular biology course. This activity introduces students to the practice of basic molecular techniques such as restriction enzyme digestion, agarose gel electrophoresis, cloning, plasmid DNA purification, Southern blotting, and sequencing. Students learn how to carry…

  12. High Throughput Micro-Well Generation of Hepatocyte Micro-Aggregates for Tissue Engineering

    NARCIS (Netherlands)

    Gevaert, Elien; Dollé, Laurent; Billiet, Thomas; Dubruel, Peter; Grunsven, van Leo; Apeldoorn, van Aart; Cornelissen, Ria

    2014-01-01

    The main challenge in hepatic tissue engineering is the fast dedifferentiation of primary hepatocytes in vitro. One successful approach to maintain hepatocyte phenotype on the longer term is the cultivation of cells as aggregates. This paper demonstrates the use of an agarose micro-well chip for the

  13. The innate pattern recognition molecule Ficolin-1 is secreted by monocytes/macrophages and is circulating in human plasma

    DEFF Research Database (Denmark)

    Honoré, Christian; Rørvig, Sara; Munthe-Fog, Lea; Hummelshøj, Tina; Madsen, Hans O; Borregaard, Niels; Garred, Peter

    2008-01-01

    and SDS-PAGE/western blot. Secretion of Ficolin-1 was investigated in cells and plasma from healthy donors through affinity purification using N-acetyl-d-glucosamine-agarose beads and ELISA. Ficolin-1 was found differentially expressed and synthesised by monocytes, macrophages and immature dendritic...

  14. One-step purification of E. coli elongation factor Tu

    DEFF Research Database (Denmark)

    Knudsen, Charlotte Rohde; Clark, Brian F. C.; Degn, B;

    1993-01-01

    The tuf A gene, encoding the E. coli elongation factor Tu, was cloned in the pGEX gene fusion system. Upon expression EF-Tu is fused to glutathione-S-transferase serving as a purification handle with affinity for glutathione immobilised on agarose. This allows purification of EF-Tu in a one...

  15. Rapid, Simple and Cost-Effective Molecular Method to Differentiate the Temperature Sensitive (ts+ MS-H Vaccine Strain and Wild-Type Mycoplasma synoviae Isolates.

    Directory of Open Access Journals (Sweden)

    Zsuzsa Kreizinger

    Full Text Available Mycoplasma synoviae infection in chickens and turkeys can cause respiratory disease, infectious synovitis and eggshell apex abnormality; thus it is an economically important pathogen. Control of M. synoviae infection comprises eradication, medication or vaccination. The differentiation of the temperature sensitive (ts+ MS-H vaccine strain from field isolates is crucial during vaccination programs. Melt-curve and agarose gel based mismatch amplification mutation assays (MAMA are provided in the present study to distinguish between the ts+ MS-H vaccine strain, its non-temperature sensitive re-isolates and wild-type M. synoviae isolates based on the single nucleotide polymorphisms at nt367 and nt629 of the obg gene. The two melt-MAMAs and the two agarose-MAMAs clearly distinguish the ts+ MS-H vaccine strain genotype from its non-temperature sensitive re-isolate genotype and wild-type M. synoviae isolate genotype, and no cross-reactions with other Mycoplasma species infecting birds occur. The sensitivity of the melt-MAMAs and agarose-MAMAs was 103 and 104 copy numbers, respectively. The assays can be performed directly on clinical samples and they can be run simultaneously at the same annealing temperature. The assays can be performed in laboratories with limited facilities, using basic real-time PCR machine or conventional thermocycler coupled with agarose gel electrophoresis. The advantages of the described assays compared with previously used methods are simplicity, sufficient sensitivity, time and cost effectiveness and specificity.

  16. Assay for the specificity of monoclonal antibodies in crossed immunoelectrophoresis

    DEFF Research Database (Denmark)

    Skjødt, K; Schou, C; Koch, C

    1984-01-01

    A method is described based on crossed immunoelectrophoresis of a complex antigen mixture in agarose gel followed by incubation of the gel with the monoclonal antibody. The bound monoclonal antibody is detected by the use of a secondary enzyme-labelled antibody. Using this technique we have been ...

  17. Digital speckle pattern interferometric measurement of diffusion coefficients in hydrogels

    Institute of Scientific and Technical Information of China (English)

    周金芳; 韩雁; 章献民; 徐坚

    2003-01-01

    The technique of real-time digital speckle pattern interferometry is proposed to study diffusion of surfactants in hydrogel. The diffusion coefficient is simply and directly determined from the interferograms. An example of diffusion coefficient measurement of surfactant in agarose gel demonstrates the usefulness of the method. The results obtained are compared with the theoretical simulating values.

  18. Composite materials having thiol groups

    International Nuclear Information System (INIS)

    A composite material having thiol groups comprises a rigid support material. The composite material may comprise a deformable gel (eg agarose) having thiol groups retained within the pore structure of a porous rigid support material (e.g. Kieselghur). The particles of composite material are used to prepare a radioactive gold isotope from a mercury 'parent' isotope. (author)

  19. In Vitro Investigations on the Toxicity and Cell Death Induced by Tamoxifen on Two Non-Breast Cancer Cell Types

    Directory of Open Access Journals (Sweden)

    S. K. Majumdar

    2001-01-01

    protein (EGFP in tamoxifen treated MEL BB-88 cells, a general feature of cells undergoing apoptosis. Tamoxifen treated cells demonstrated internucleosomal damages of the genomic DNA and DNA fragmentations, evidenced by an increase in free nucleosomes, and distinctive DNA smear patterns on the agarose gel.

  20. Three-dimensional imaging analysis of Yersinia ruckeri infected rainbow trout (Oncorhynchus mykiss) gills by optical projection tomography

    DEFF Research Database (Denmark)

    Otani, Maki; Raida, Martin Kristian

    incubated whole with rabbit anti-Y. ruckeri polyclonal antibody and Alexa Fluor®594 conjugated goat anti-rabbit IgG. After embedding in 1% low melting point agarose, specimens were dehydrated in 100% methanol and cleared in BABB (benzyl alcohol: benzyl benzoate) for OPT scanning. 3D imaging results showed...

  1. Characterizing of Cooling Equipment for Closed Greenhouses

    NARCIS (Netherlands)

    Zwart, de H.F.; Kempkes, F.L.K.

    2008-01-01

    In order to develop new eco-sustainable technologies to set up biodegradable films for agricultural activities, spray mulching coating have been planned, prepared and tested on experimental fields. The suitable polymers used to this purpose were Arabic Gums and Agarose. Cellulose fibres were added t

  2. The Influences of LuxX in "Escherichia Coli" Biofilm Formation and Improving Teacher Quality through the Bio-Bus Program

    Science.gov (United States)

    Robbins, Chandan Morris

    2012-01-01

    The objectives of this work are: (1) to agarose-stabilize fragile biofilms for quantitative structure analysis; (2) to understand the influences of LuxS on biofilm formation; (3) to improve teacher quality by preparing Georgia's middle school science teachers to integrate inquiry-based, hands-on research modules in the classroom. Quantitative…

  3. An improved cell recovery method for iron oxidizing bacterial (IOB) enrichments

    DEFF Research Database (Denmark)

    Yu, Ran; Graf, Joerg; Smets, Barth F.

    2008-01-01

    Two cell recovery methods for IOB enrichments were evaluated for DNA extraction and further PCR-based 16S rRNA gene clone library creation. One was a published method consisting of heating plus oxalic acid treatment and the other one was a new method based on enzymatic agarose digestion (using β...

  4. Micromachining on copper and nickel by electrochemical wet stamping

    International Nuclear Information System (INIS)

    The fabrication of microstructures on copper and nickel has been achieved by an approach named electrochemical wet stamping (E-WETS). The E-WETS process allows the direct imprinting of microscopic reliefs on an agarose stamp into nickel and copper through a selective anodic dissolution process. The pre-patterned agarose with a high gel strength that has been soaked in a desired etching solution is employed as a stamp. It can direct and supply the solution preferentially on the contact area owing to the constant supply of electrolyte from the agarose stamp to the interface and then the electrochemical reaction is limited only to the contact area. Simultaneously, the product can be removed from the gel/substrate interface. On the basis of the electrochemical behavior of the copper electrode in contact with the agarose full of 0.1 M HClO4, the potential for the electrochemical micromachining was chosen to be a relatively low value, 0.4 V versus SCE, to prevent the generation of cuprous oxide. A voltage pulse was applied to the electrochemical machining of nickel. The pulse amplitude was set to 8 V versus SCE to take the electrode into the transpassivation region rather than the passivation region in the pulse duration. Furthermore, the different electrochemical mechanisms involved in the etching process have been discussed in detail

  5. Directing functional chemistries on micropatterned conducting polymers for all-polymer cell analysis microsystems

    DEFF Research Database (Denmark)

    Lind, Johan Ulrik; Daugaard, Anders Egede; Andresen, Thomas Lars;

    Micrometer scale electrical circuits of PEDOT (poly(3,4-dioxythiophene)) were created by locally oxidizing PEDOT thin films with an agarose stamp containing the oxidizing agent NaOCl. The oxidized PEDOT was removed completely by applying detergents. The process was sufficiently mild that chemical...

  6. Kinetic resolution of racemic mixtures in gel media

    Science.gov (United States)

    Petrova, Rositza Iordanova

    The goal of this research was to investigate the effect of chiral gels on the chiral crystal nucleation and growth and assess the gels' potential as media for kinetic separation of racemic mixtures. The morphologies of asparagine monohydrate and sodium bromate crystals grown in different gel media were examined in order to discern the effect of gel structure and density on the relative growth rates of those materials. Different crystal habits were observed when the gel chemical composition, density and solute concentration were varied. These studies showed that the physical properties of the gel, such as gel density and pore size, as well as its chemical composition affect the crystal habit. The method of kinetic resolution in gel media was first applied to sodium chlorate, which is achiral in solution but crystallizes in a chiral space group. Crystallization in agarose gels yielded an enantiomorphic bias, the direction and magnitude of which could be affected by changing the temperature or by the addition of an achiral cosolvent. Aqueous gels at 6°C produced crystalline mixtures enriched with the d-enantiomorph, while crystallization under MeOH diffusion favored l-crystals. Optimized conditions yielded e.e. of 53% of l-enantiomorph. The method was next applied to the organic molecular crystals of asparagine monohydrate and threonine. Asparagine monohydrate growth in aqueous agarose and iota-carrageenan gels produced crystal mixtures enriched with D-enantiomer. The degree of resolution was higher when the total amount of asparagine crystallized was low. The success of the resolution depends strongly on the concentrations of solute and the geling substance. Growth from agarose gels yielded e.e. of 44% under optimized conditions. The same method was applied to the resolution of Thr, albeit with modest success. In an effort to improve the resolution of asparagine monohydrate, agarose was synthetically modified by esterifying its side chains with homochiral asparagyl

  7. Diffusion measurement in ferrous infused gel dosimeters

    International Nuclear Information System (INIS)

    Background: The compositions of Ferrous sulphate, Agarose and Xylenol orange dye and Ferrous sulphate, Gelatin and Xylenol orange dye in solution of distilled water and sulphuric acid are two tissue-equivalent gel dosimeters. Ionizing radiation causes oxidation of Fe2+ ion to Fe3+ ions which diffuse through the gel matrix and blur the image of absorbed dose over a period of hours after irradiation. Materials and methods: 25 m M sulphuric acid, 0.4 mm ferrous ammonium sulphate, 0.2 mm xylenol orange dye and 1% by weight agarose in distilled water named Agarose and Xylenol orange dye and 0.1 mm ferrous ammonium sulphate, 0.1 mm xylenol orange dye, 50 mm sulphuric acid and 5% by weight gelatin in distilled water named Gelatin and Xylenol orange dye are used as two gel dosimeters. All chemicals were supplied by Sigma Ald ridge Company, Germany. The gels were poured in Perspex casts and were irradiated to a beam of X ray from linear accelerators or X ray machine. Results: In this study diffusion coefficients of Agarose and Xylenol orange dye and Gelatin and Xylenol orange dye dosimeters have been measured through a computer program for different temperature. The ferric ion diffusion coefficient (D) for the Agarose and Xylenol orange dye and Gelatin and Xylenol orange dye dosimeters were measured as (1.19.±0.03) x 10-2 cm2.hr -1 and (0.83±0.03) x 10-2 cm2.hr-1 respectively at room temperature. Conclusion: For both dosimeters the diffusion coefficients decreased with gel storage temperatures down to 6 digC. Gelatin and Xylenol orange dye dosimeters have advantage of lower diffusion coefficient for a specified temperature

  8. Substrate recognition and hydrolysis by a family 50 exo-β-agarase, Aga50D, from the marine bacterium Saccharophagus degradans.

    Science.gov (United States)

    Pluvinage, Benjamin; Hehemann, Jan-Hendrik; Boraston, Alisdair B

    2013-09-27

    The bacteria that metabolize agarose use multiple enzymes of complementary specificities to hydrolyze the glycosidic linkages in agarose, a linear polymer comprising the repeating disaccharide subunit of neoagarobiose (3,6-anhydro-l-galactose-α-(1,3)-d-galactose) that are β-(1,4)-linked. Here we present the crystal structure of a glycoside hydrolase family 50 exo-β-agarase, Aga50D, from the marine microbe Saccharophagus degradans. This enzyme catalyzes a critical step in the metabolism of agarose by S. degradans through cleaving agarose oligomers into neoagarobiose products that can be further processed into monomers. The crystal structure of Aga50D to 1.9 Å resolution reveals a (β/α)8-barrel fold that is elaborated with a β-sandwich domain and extensive loops. The structures of catalytically inactivated Aga50D in complex with non-hydrolyzed neoagarotetraose (2.05 Å resolution) and neoagarooctaose (2.30 Å resolution) provide views of Michaelis complexes for a β-agarase. In these structures, the d-galactose residue in the -1 subsite is distorted into a (1)S3 skew boat conformation. The relative positioning of the putative catalytic residues are most consistent with a retaining catalytic mechanism. Additionally, the neoagarooctaose complex showed that this extended substrate made substantial interactions with the β-sandwich domain, which resembles a carbohydrate-binding module, thus creating additional plus (+) subsites and funneling the polymeric substrate through the tunnel-shaped active site. A synthesis of these results in combination with an additional neoagarobiose product complex suggests a potential exo-processive mode of action of Aga50D on the agarose double helix. PMID:23921382

  9. MR thermometry for laser-induced thermotherapy at 1.5 tesla; MR-Thermometrie bei 1,5 Tesla zur thermischen Ablation mittels laserinduzierter Thermotherapie

    Energy Technology Data Exchange (ETDEWEB)

    Meister, D.; Huebner, F.; Mack, M.; Vogl, T.J. [Frankfurt Univ. (Germany). Inst. fuer Diagnostische und Interventionelle Radiologie

    2007-05-15

    Purpose: Evaluation of thermometry with fast MR sequences for laser-induced interstitial laser therapy (LITT) and verification of the thermometric results with a fiber-optic thermometer. Method and Materials: In vitro experiments were conducted using an agarose gel mixture and pig liver lobes. MR-guided LITT was performed using a laser power between 3 and 15?watts. Thermometry was performed using longitudinal relaxation time T1 and proton resonance frequency shift (PRF) methods under acquisition of amplitude and phase shift images. PRF was measured with a fast spoiled GRE sequence. Four different sequences were used for T1 thermometry: gradient echo (GE), TrueFISP (TRUFI), Saturation Recovery Turbo-FLASH (SRTF) and Inversion Recovery Turbo-FLASH (IRTF) sequences. The temperature was controlled using a fiber-optic Luxtron device and correlated with the MR temperature. The range of applied and monitored temperatures exceeded 80 degrees Celsius. Results: The temperature dependence showed a good linear relationship up to 60 degrees Celsius. Calibration experiments for the T1 method delivered coefficients of determination from 0.977 to 0.997 for agarose and from 0.958 to 0.995 for the pig liver samples. The IRTF sequence had the highest temperature sensitivity (agarose 0.99, liver 1.19). During LITT the TRUE-FISP sequence exhibited a strong nonlinear relationship. R{sup 2} of this sequence was 0.809 in the agarose experiments. The average temperature errors when heated up to 80 degrees Celsius were 3.86 - 11.38 degrees Celsius for Agarose gel and 5.7 - 12.16 degrees Celsius for the liver tissue. SRTF and IRTF sequences exhibited the most linear relationship with temperature but were more dependent on tissue differences. (orig.)

  10. Electron beam sterilization of the Agaroze gel used for electrophoresis

    International Nuclear Information System (INIS)

    Electron beam sterilization is based on its ability to kill pathogenic microorganisms. It is applied to a broad range of disposable medical products such as syringes, needles, surgical sutures, transplant kits, inhalation and dialysis equipment, blood-handling equipment, wound and burn dressings, gloves, masks, gowns, Petri dishes and pipettes. By this work we intend to extend the EB irradiation to the sterilization of the agarose gel put on plastic plates. Electrophoresis of serum proteins on agarose gel is an important investigation method of variety disproteinemia types, used in clinical laboratory. By this method are separated six proteic fractions: Albumin; Alpha 1 globulin consisting of α1 lipoproteins and α1 antitrypsin; Alpha 2 globulin consisting of α2 macroglobulin and haptoglobin; Beta 1 globulin consisting of transferrin and β lipoprotein; Beta 2 globulin consisting of complement C3; Gamma globulin consisting of immunoglobulins and fibrogen. Agarose is a natural colloid extracted from seaweed. It is very fragile and easily destroyed by handling. Agarose gel can be processed faster than polyacrylamide gels, but the agarose gel is a favorable medium for the microorganisms' development, which is the cause for the degradation of the gel and of the results. Thus, the sterilization of the plates with agarose gels becomes important. A disadvantage is that ionizing radiation degrades some plastic gels above a certain absorbed dose level. In view of this important aspects the sterilization of the agarose gel, which is put on plastic plates, by electron beam irradiation has been studied. Also, the effects of the electron beam irradiation upon the agarose gel and on the process of the proteic fraction separation have been investigated. In the experimental studies are used the plastic plates with 1% agarose gel in Tris-barbital tampon, supplied by DDS DIAGNOSTIC-Romania. The used migration solution is Tris-barbital tampon pH 8.6. In order to establish the

  11. Reptation dynamics of single-walled carbon nanotubes in a permanent network

    Science.gov (United States)

    Fakhri, Nikta; Mackintosh, Fred; Cognet, Laurent; Lounis, Brahim; Pasquali, Matteo

    2010-03-01

    Single-walled carbon nanotubes (SWCNTs) are an ideal system of semiflexible filaments with tunable bending stiffness. By exploiting their near-infrared fluorescence, we image directly the motion of SWCNTs in a network (agarose gel). We determine the SWCNT diameter (and bending stiffness) spectroscopically, and we control the network pore size by changing the agarose concentration. Image analysis shows clearly that SWCNTs move by reptation through the pore network. We quantify the dependence of SWCNTs mobility on SWCNT bending stiffness, length and pore sizes. Our results show conclusively that, even when the SWCNT length is much smaller than the persistence length, the flexibility of filaments enhances rotational diffusion. These results confirm earlier predictions of Odijk (1983), and show that the Doi-Edwards scaling fails to capture the filaments' motion. This study provides a fundamental understanding of reptation dynamics of semiflexible filaments.

  12. N-Terminal methionine processing by the zinc-activated Plasmodium falciparum methionine aminopeptidase 1b.

    Science.gov (United States)

    Calcagno, Sarah; Klein, Christian D

    2016-08-01

    The methionine aminopeptidase 1b from Plasmodium falciparum (PfMetAP 1b) was cloned, expressed in Escherichia coli and characterized. Surprisingly, and in contrast to other methionine aminopeptidases (MetAPs) that require heavy-metal cofactors such as cobalt, the enzyme is reliably activated by zinc ions. Immobilization of the enzyme is possible by His-tag metal chelation to iminodiacetic acid-agarose and by covalent binding to chloroacetamido-hexyl-agarose. The covalently immobilized enzyme shows long-term stability, allowing a continuous, heterogenous processing of N-terminal methionines, for example, in recombinant proteins. Activation by zinc, instead of cobalt as for other MetAPs, avoids the introduction of heavy metals with toxicological liabilities and oxidative potential into biotechnological processes. The PfMetAP 1b therefore represents a useful tool for the enzymatic, posttranslational processing of recombinant proteins. PMID:27023914

  13. Study of calf thymus DNA irradiated in vitro with MeV fluorine ions

    International Nuclear Information System (INIS)

    A study of the fragments of DNA irradiated with MeV ions is important for the understanding of the DNA damage mechanism and the subsequent biological effects (induced by heavy ions). In this experiment, the products of calf thymus DNA (CT DNA) irradiated with MeV fluorine ions were analyzed using agarose gel electrophoresis, modified time-of-flight mass spectrometer (MALDI-TOF), and high-performance liquid chromatography (HPLC). The results showed that the molecular mass of the fragments were concentrated around 831 bp with agarose gel electrophoresis, there was no observable product in the range of 1,000-30,000 (m/q) using MALDI-TOF, and small biomolecules were separated from the products. The results of this study indicated that the strand breaks of calf thymus DNA induced by MeV fluorine ions were nonrandom. (authors)

  14. Aromatic proteinaceous surfactants stabilize long-lived gas microbubbles from natural sources

    Science.gov (United States)

    Darrigo, J. S.

    1981-01-01

    Three different types of protein-specific chemical tests were performed on long-lived gas microbubbles derived from aqueous solutions of agarose powder and from filtered aqueous extracts of Hawaiian forest soil. The separate protein-specific tests involved use of either 0.3% (w/v) ninhydrin, 100 microM methylene blue dye, or 0.7-1.0 M 2-hydroxy-5-nitrobenzyl bromide. The chemical test results obtained with each of the two natural substances, i.e., agarose powder and Hawaiian forest soil, were very similar and indicate that the biological surfactants which surround and stabilize long-lived gas microbubbles are proteinaceous compounds that contain, and whose surface activity depends upon, aromatic amino acid residues, particularly tryptophan.

  15. Analyse statistique de données d'écologie microbienne

    OpenAIRE

    Le Duot, A.

    2010-01-01

    / L'objectif principal de ce stage était donc l'adaptation et l'utilisation des outils d'analyse des profils d'ADN obtenus sur gel d'agarose et par SSCP au travers de deux études : 1. la première, basée sur des profils obtenus par électrophorèse sur gel d'agarose, avait pour objectif d'évaluer la persistance de souches résistantes aux antibiotiques dans l'environnement lors de l'épandage de fumier de volailles. En effet, l'épandage des effluents d'élevage comporte des risques pour l'enviro...

  16. QNS measurements of water in biological and model systems

    Energy Technology Data Exchange (ETDEWEB)

    Trantham, E.C.; Rorschach, H.E.; Clegg, J.C.; Hazlewood, C.F.; Nicklow, R.M.

    1982-01-01

    Results are presented on the quasi-elastic spectra of 0.95 THz neutrons scattered from pure water, a 20% agarose gel and cysts of the brine shrimp (Artemia) of hydration 1.2 gms H/sub 2/O per gm of dry solids. The lines are interpreted with a two-component model in which the hydration water scatters elastically and the free water is described by a jump-diffusion correlation function. The results for the line widths GAMMA(Q/sup 2/) are in good agreement with previous measurements for the water sample but show deviations from pure water at large Q for agarose and the Artemia cysts that suggest an increased value of the residence time in the jump-diffusion model.

  17. QNS measurements on water in biological and model systems

    Energy Technology Data Exchange (ETDEWEB)

    Trantham, E.C.; Rorschach, H.E.; Clegg, J.C.; Hazlewood, C.F.; Nicklow, R.M.

    1981-01-01

    Results are presented on the quasi-elastic spectra of 0.95 THz neutrons scattered from pure water, a 20% agarose gel and cysts of the brine shrimp (Artemia) of hydration 1.2 gms H/sub 2/O per gm of dry solids. The lines are interpreted with a two-component model in which the hydration water scatters elastically and the free water is described by a jump-diffusion correlation function. The results for the line widths GAMMA(Q/sup 2/) are in good agreement with previous measurements for the water sample but show deviations from pure water at large Q for agarose and the Artemia cysts that suggest an increased value of the residence time in the jump-diffusion model.

  18. Comparison between pulsed-field and constant-field gel electrophoresis for measurement of DNA double-strand breaks in irradiated Chinese hamster ovary cells

    International Nuclear Information System (INIS)

    Pulsed-field gel electrophoresis (PFGE) is one of the most sensitive methods for detecting DNA double-strand breaks in mammalian cells. However, it has been observed that constant-field gel electrophoresis (CFGE), when optimized, can detect breaks with equal efficiency. The migration of DNA from the well and the separation of DNA molecules according to size appear to be different processes; only the latter requires the application of PFGE. CFGE is very sensitive and can detect DNA damage produced by less than 5Gy of radiation. Low voltage (ca.0.6V/cm) during electrophoresis appears to be essential for the migration of the largest fraction of DNA from the agarose plug containing the cells; the electrophoresis run time, cell density in the plug, agarose concentration, nature of detergent and extent of radiolabelling are less important. It is concluded that CFGE is equally sensitive but more rapid and economical than PFGE for the measurement of DNA damage. (author)

  19. Molecular diversity of snake venom nerve growth factors.

    Science.gov (United States)

    Trummal, Katrin; Tõnismägi, Külli; Paalme, Viiu; Järvekülg, Lilian; Siigur, Jüri; Siigur, Ene

    2011-09-15

    Nerve growth factor (NGF) is a protein which stimulates the differentiation and maintenance of sympathetic and embryonic sensory neurons. Snake venoms are a rich source of NGF. Due to small quantities it is sometimes difficult and laborious to isolate NGF from the venoms. In this study the use of Ni-NTA-agarose for isolation of NGF is studied. Anti-Vipera lebetina NGF antibodies were used for identification of NGF during Ni-NTA-agarose fractionation as well as for cross-reaction studies with 21 snake venoms. All studied venoms contained NGF. The molecular masses of the NGFs from Echis ocellatus, Agkistrodon contortrix contortrix, A. bilineatus, A. blomhoffii, A. saxatilis, Calloselasma rhodostoma, Bothrops jararaca and B. lanceolatus were determined for the first time. Some previous results of the NGF studies are revaluated. PMID:21801740

  20. Production of agaro- and carra-oligosaccharides by partial acid hydrolysis of galactans

    Directory of Open Access Journals (Sweden)

    Diogo R. B. Ducatti

    2011-04-01

    Full Text Available Agaro- and carra-oligosaccharides were produced by partial acid hydrolysis of commercial agarose and kappa-carrageenan. Di- and tetrasaccharides were purified by gel filtration chromatography and characterized by NMR (1D and 2D spectroscopy and ESIMS. The following oligosaccharides were obtained: agarobiose, agarotetraose, kappa-carrabiose and kappa-carratetraose. Agarobiose and agarotetraose were used as standards to develop a high performance size exclusion chromatography (HPSEC method which was utilized to study the hydrolysis rate of agarose and oligosaccharide production. Six hours of hydrolysis (0.1 M TFA, 65 ºC produced mainly di- and tetrasaccharides. The methodology for oligosaccharide production and evaluation developed in the present work shows good potential for the production of bioactive oligosaccharides.

  1. Restriction site detection in repetitive nuclear DNA sequences of Trypanosoma evansi for strain differentiation among different isolates.

    Science.gov (United States)

    Shyma, K P; Gupta, S K; Gupta, J P; Singh, Ajit; Chaudhari, S S; Singh, Veer

    2016-09-01

    The differences or similarities among different isolates of Trypanosoma evansi through endonuclease profile was identified in the present study. The repetitive nuclear DNA of T. evansi isolated from infected cattle, buffalo and equine blood was initially amplified by PCR using specific primers. A panel of restriction enzymes, EcoRI, Eco91l, HindIII and PstI were for complete digestion of PCR products. Agarose gel electrophoresis of digested product did not show cleavage fragments and only single DNA band of the original size was visible in the ethidium bromide stained agarose gel. This indicated that the 227 bp PCR product from repetitive sequence had no site-specific cleavage sites for the REs used in this study. No heterogeneity in the repetitive nuclear DNA restriction endonuclease profile among the different isolates was recorded. PMID:27605842

  2. Further Development of Scaffolds for Regeneration of Nerves

    Science.gov (United States)

    Sakamoto, Jeffrey; Tuszynski, Mark

    2009-01-01

    Progress has been made in continuing research on scaffolds for the guided growth of nerves to replace damaged ones. The scaffolds contain pores that are approximately cylindrical and parallel, with nearly uniform widths ranging from tens to hundreds of microns. At the earlier stage of development, experimental scaffolds had been made from agarose hydrogel. Such a scaffold was made in a multistep process in which poly(methyl methacrylate) [PMMA] fibers were used as templates for the pores. The process included placement of a bundle of the PMMA fibers in a tube, filling the interstices in the tube with a hot agarose solution, cooling to turn the solution into a gel, and then immersion in acetone to dissolve the PMMA fibers. The scaffolds were typically limited to about 25 pores per scaffold, square cross sections of no more than about 1.5 by 1.5 mm, and lengths of no more than about 2 mm.

  3. QNS measurements of water in biological and model systems

    International Nuclear Information System (INIS)

    Results are presented on the quasi-elastic spectra of 0.95 THz neutrons scattered from pure water, a 20% agarose gel and cysts of the brine shrimp (Artemia) of hydration 1.2 gms H2O per gm of dry solids. The lines are interpreted with a two-component model in which the hydration water scatters elastically and the free water is described by a jump-diffusion correlation function. The results for the line widths GAMMA(Q2) are in good agreement with previous measurements for the water sample but show deviations from pure water at large Q for agarose and the Artemia cysts that suggest an increased value of the residence time in the jump-diffusion model

  4. QNS measurements on water in biological and model systems

    International Nuclear Information System (INIS)

    Results are presented on the quasi-elastic spectra of 0.95 THz neutrons scattered from pure water, a 20% agarose gel and cysts of the brine shrimp (Artemia) of hydration 1.2 gms H2O per gm of dry solids. The lines are interpreted with a two-component model in which the hydration water scatters elastically and the free water is described by a jump-diffusion correlation function. The results for the line widths GAMMA(Q2) are in good agreement with previous measurements for the water sample but show deviations from pure water at large Q for agarose and the Artemia cysts that suggest an increased value of the residence time in the jump-diffusion model

  5. Study on CT DNA in vitro irradiated with MeV fluorine ions

    International Nuclear Information System (INIS)

    The study on products of CT DNA irradiated with heavy ion is very important to understand the biologic effect induced by heavy ions. The authors have analyzed the products of CT DNA irradiated with MeV F ions using agarose gel electrophoresis, MALDI-TOF and HPLC methods. The results show that the products are mostly multi-DSB in our experiment observed by agarose gel electrophoresis, and there are not observable products between 1,000 and 30,000 (m/z) using MALDI-TOF method. HPLC result indicates some small biomolecules occurring in the products. These results suggest that the break of CT DNA induced by MeV heavy ions irradiation was not a random process. (authors)

  6. Quantification of DNA damage by single-cell electrophoresis

    International Nuclear Information System (INIS)

    A simple technique of micro-agarose gel electrophoresis has been developed to quantify DNA damage in individual cells. Cells are embedded in agarose gel on microscope slides, lysed by detergents and then electrophoresed for a short time under neutral or alkaline condition. In irradiated cells, DNA migrates from the nucleus toward the anode, displaying commet-like pattern by staining with DNA-specific fluorescence dye. DNA damage is evaluated by measuring the distance of DNA migration. The technique was applied for measuring DNA damage in single cells exposed to 60Co γ-rays, or to KUR radiation in the presence or absence of 10B-enriched boric acid. The enhanced production of double-stranded DNA breaks by 10B(n,α)7Li reaction was demonstrated here. The significant increase in the length of DNA migration was observed in single cells exposed to such a low dose as 20 cGy after alkaline micro electrophoresis. (author)

  7. Determination of the cationic amphiphilic drug-DNA binding mode and DNA-assisted fluorescence resonance energy transfer amplification

    Science.gov (United States)

    Yaseen, Zahid; Banday, Abdul Rouf; Hussain, Mohammed Aamir; Tabish, Mohammad; Kabir-ud-Din

    2014-03-01

    Understanding the mechanism of drug-DNA binding is crucial for predicting the potential genotoxicity of drugs. Agarose gel electrophoresis, absorption, steady state fluorescence, and circular dichroism have been used in exploring the interaction of cationic amphiphilic drugs (CADs) such as amitriptyline hydrochloride (AMT), imipramine hydrochloride (IMP), and promethazine hydrochloride (PMT) with calf thymus or pUC19 DNA. Agarose gel electrophoresis assay, along with absorption and steady state fluorescence studies, reveal interaction between the CADs and DNA. A comparative study of the drugs with respect to the effect of urea, iodide induced quenching, and ethidium bromide (EB) exclusion assay reflects binding of CADs to the DNA primarily in an intercalative fashion. Circular dichroism data also support the intercalative mode of binding. Besides quenching, there is fluorescence exchange energy transfer (FRET) in between CADs and EB using DNA as a template.

  8. Partial characterization of the N-linked oligosaccharide structures on Pselectin glycoprotein ligand-1 (PSGL-1)

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    PSGL-1,a specific ligand for P-,E- and L-selectin,was isolated from in vivo [3H]-glucosamine labeled HL-60 cells by a combination of wheat germ agglutinin-agarose and P- or E-selectin-agarose chromatography.N-linked oligosaccharides were released from the purified,denatured ligand molecule by peptide: N-glycosidase F treatment and,following separation by Sephacryl S-200 chromatography,partially characterized using lectin,ion-exchange and size-exclusion chromatography in combination with glycosidase digestions.The data obtained suggest that the N-glycans on PSGL-1 are predominantly core-fucosylated,multiantennary complex type structures with extended,poly-N-acetyllactosamine containing outer chains.A portion of the outer chains appears to be substituted with fucose indicating that the N-glycans,in addition to the O-glycans on PSGL-1,may be involved in selectin binding.

  9. REE bound DNA in natural plant

    Institute of Scientific and Technical Information of China (English)

    王玉琦; 江平; 郭繁清; 张智勇; 孙景信; 许雷; 曹国印

    1999-01-01

    The binding of rare earth elements (REEs) with nucleic acids in the leaves of fern Dicranopteris dichotoma (DD) has been studied by molecular activation analysis (MAA). The REEs bound DNA (REE-DNA) was obtained from the leaves of DD. The CTAB-based procedure was modified for extraction of total DNA. The purity of DNA was examined by UV spectroscopy. The DNA obtained was separated and determined by agarose gel electrophoresis further. Meanwhile, the contents of eight rare earth elements (La, Ce, Nd, Sm, Eu,Tb, Yb and Lu) in REE-DNA were detected by instrumental neutron activation analysis (INAA). The results showed that REE-DNA with higher purity could be extracted from plant using this method. It was also found that REEs were bound firmly with DNA in the leaves of DD. The molecular weight (MW) of REE-DNA band was about 22 kb in agarose gel electrophoresis.

  10. Dynamic changes of apoptosis in duck embryo fibroblasts induced by new type Gosling viral enteritis virus

    Institute of Scientific and Technical Information of China (English)

    Shun Chen; Anchun Cheng; Mingshu Wang; Xiaoyue Chen

    2008-01-01

    The monolayer duck embryo fibroblast (DEF) cells were experimentally infected with new type Gosling viral enteritis virus (NGVEV) and the dynamic changes of apoptosis were detected at different time points after NGVEV infection by transmission electron microscopy (TEM), DNA agarose gel electrophoresis and Annexin V-FITC/PI stained fluorescence-activated cell sorter (FACS). The result shows that NGVEV can induce infected cells undergoing apoptosis and changing regularly. A series of characteristic apoptotic morphological changes including shrinkage of the cells, chromatin condensation and margination, as well as formation of apoptotic bodies, wereobserved by TEM. The typical ladder pattern of DNA fragmentation was demonstrated by agarose gel electrophoresis. And using flow cytometry analysis of Annexin V-FITC/PI staining, the dead, viable, apoptotic and necrotic cells could be analyzed quantitatively.

  11. Study of calf thymus DNA irradiated in vitro with MeV fluorine ions

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    A study of the fragments of DNA irradiated with MeV ions is important for the understanding of the DNA damage mechanism and the subsequent biological effects (induced by heavy ions). In this experiment, the products of calf thymus DNA (CT DNA) irradiated with MeV fluorine ions were analyzed using agarose gel electrophoresis,modified time-of-flight mass spectrometer (MALDI-TOF), and high-performance liquid chromatography (HPLC).The results showed that the molecular mass of the fragments were concentrated around 831 bp with agarose gel electrophoresis, there was no observable product in the range of 1,000- 30,000 (m/q) using MALDI-TOF, and small biomolecules were separated from the products. The results of this study indicated that the strand breaks of calf thymus DNA induced by MeV fluorine ions were nonrandom.

  12. Optimization of the southern electrophoretic transfer method

    International Nuclear Information System (INIS)

    The technique of separating DNA fragments using agarose gel electrophoresis is essential in the analysis of nucleic acids. Further, after the method of transferring specific DNA fragments from those agarose gels to cellulose nitrate membranes was developed in 1975, a method was developed to transfer DNA, RNA, protein and ribonucleoprotein particles from various gels onto diazobenzyloxymethyl (DBM) paper using electrophoresis as well. This paper describes the optimum conditions for quantitative electrophoretic transfer of DNA onto nylon membranes. This method exemplifies the ability to hybridize the membrane more than once with specific RNA probes by providing sufficient retention of the DNA. Furthermore, the intrinsic properties of the nylon membrane allow for an increase in the efficiency and resolution of transfer while using somewhat harsh alkaline conditions. The use of alkaline conditions is of critical importance since we can now denature the DNA during transfer and thus only a short pre-treatment in acid is required for depurination. 9 refs., 7 figs

  13. An Immunoabsorption Column Conifguration for Hepatitis B Virus

    Institute of Scientific and Technical Information of China (English)

    2013-01-01

    Objective To conifgure an immunoabsorption column for hepatitis B virus. Methods Being activated by epichlorohydrin, the human antibody HBsAb-IgG was bound to the carrier of agarose gel. The configuration process was as follows: the synthesis of epoxide matrix, the synthesis and activation of amino matrix, the synthesis of aldehydic matrix, the synthesis of immunoabsorption matrix, the end capping and reduction of unbound aldehydic, the blocking of unbound mass and the iflling of the column. Results The bound rate of activated agarose gel and antibody HBsAb-IgG is 85.07%. By plasma adsorption experiment, it is revealed that the immunoabsorption column can absorb and eliminate 58.97%of HBsAg and 53.1%of hepatitis B virus particles in extracorporeal plasma. Conclusions The immunoabsorption column for hepatitis B virus can absorb and eliminate HBsAg and hepatitis B virus particles in extracorporeal plasma.

  14. Light deflection and convection in diffusion experiments using holographic interferometry

    International Nuclear Information System (INIS)

    A study of the effect of light deflection during diffusion studies of ethanol into agarose gel using holographic laser interferometry is presented. Furthermore it also demonstrates how a diffusive flux could give rise to a convective flux in holographic laser interferometry experiments. The convective and diffusive mass transfer is also theoretically compared in both a liquid phase and a gel phase for the ethanol-agarose system used. The current study shows that errors due to light deflection in holographic laser interferometry are extremely small and can be neglected. It also shows the importance of designing the diffusion experiments to avoid natural convection. In gels the convective flow is cancelled by the friction forces between the liquid and the polymer network. However, in the liquid phase the natural convection could occur even though the density differences in the phase are small. (author)

  15. Pulsed-Field Electrophoresis: Application of a Computer Model to the Separation of Large DNA Molecules

    Science.gov (United States)

    Lalande, Marc; Noolandi, Jaan; Turmel, Chantal; Rousseau, Jean; Slater, Gary W.

    1987-11-01

    The biased reptation theory has been applied to the pulsed-field electrophoresis of DNA in agarose gels. A computer simulation of the theoretical model that calculates the mobility of large DNA molecules as a function of agarose pore size, DNA chain properties, and electric field conditions has been used to generate mobility curves for DNA molecules in the size range of the larger yeast chromosomes. Pulsed-field electrophoresis experiments resulting in the establishment of an electrophoretic karyotype for yeast, where the mobility of the DNA fragments is a monotonic function of molecular size for the entire size range that is resolved (200-2200 kilobase pairs), has been compared to the theoretical mobility curves generated by the computer model. The various physical mechanisms and experimental conditions responsible for band inversion and improved electrophoretic separation are identified and discussed in the framework of the model.

  16. Electrophoresis and electro-affinity transfer with specific antibodies to alpha-fetoprotein for detection of circulating immune complexes of alpha-fetoprotein.

    Directory of Open Access Journals (Sweden)

    Taketa,Kazuhisa

    1984-08-01

    Full Text Available A combination of agarose gel electrophoresis and a newly developed technique of electro-affinity transfer was applied to the detection of circulating immune complexes of human alpha-fetoprotein (AFP and anti-AFP. After electrophoretic transfer to nitrocellulose membrane, to which affinity-purified polyclonal horse antibodies to human AFP were bound, the membranes were treated with or without rabbit immunoglobulins to human AFP, followed by overlaying with horseradish peroxidase-labeled goat anti-rabbit IgG for color development. Artificial complexes formed in vitro from human AFP and rabbit anti-AFP were clearly separated from free AFP by the agarose electrophoresis. The complexes were stained 20-40% as dark as the equivalent amount of free AFP by treatment with rabbit anti-AFP, and 10-20% as dark without the antibody treatment over a wide range of antigen-antibody ratios.

  17. Macrophage uptake of cylindrical microparticles investigated with correlative microscopy.

    OpenAIRE

    Tscheka, Clemens; Hittinger, Marius; Lehr, Claus-Michael; Schneider-Daum, Nicole; Schneider, Marc

    2015-01-01

    Cylindrical particles offer the opportunity to develop controlled and sustained release systems for the respiratory tract. One reason is that macrophages can phagocyte such particles only from either of the two ends. We investigated the uptake behaviour of murine alveolar macrophages incubated with elongated submicron-structured particles. For that purpose, fluorescent model silica nanoparticles were interconnected with the biocompatible polysaccharide agarose, building up cylindrical particl...

  18. Bacterial Plasmids in Antarctic Natural Microbial Assemblages

    OpenAIRE

    Kobori, Hiromi; Sullivan, Cornelius W.; Shizuya, Hiroaki

    1984-01-01

    Samples of psychrophilic and psychrotrophic bacteria were collected from sea ice, seawater, sediments, and benthic or ice-associated animals in McMurdo Sound, Antarctica. A total of 155 strains were isolated and tested for the presence of plasmids by DNA agarose gel electrophoresis. Thirty-one percent of the isolates carried at least one kind of plasmid. Bacterial isolates taken from sediments showed the highest plasmid incidence (42%), and isolates from seawater showed the lowest plasmid inc...

  19. Purification and characterization of four proteases from a clinical isolate of Serratia marcescens kums 3958.

    OpenAIRE

    Matsumoto, K; Maeda, H.; Takata, K; Kamata, R; Okamura, R

    1984-01-01

    Four distinct proteases were purified to homogeneity from culture filtrates of Serratia marcescens kums 3958, a fresh isolate from a patient with a severe corneal ulcer. Purification was achieved by ammonium sulfate precipitation, DEAE-cellulose ion-exchange chromatography, and Sephadex gel filtration chromatography. The proteases were differentiated from each other by polyacrylamide gel electrophoresis with or without sodium dodecyl sulfate and by immunodiffusion in agarose gels. The molecul...

  20. Identification of Cysteine Proteases and Screening of Cysteine Protease Inhibitors in Biological Samples by a Two-Dimensional Gel System of Zymography and Reverse Zymography

    OpenAIRE

    Eiichi Saitoh; Shinya Yamamoto; Eishiro Okamoto; Yoshimi Hayakawa; Takashi Hoshino; Ritsuko Sato; Satoko Isemura; Sadami Ohtsubo; Masayuki Taniguchi

    2007-01-01

    We have developed a two-dimensional (2D-) gel system of zymography and reverse zymography for the detection and characterization of proteases and protease inhibitors. Isoelectric focusing (IEF) agarose gels with pH gradients were employed for separation in the first-dimension and sodium dodecyl sulfate (SDS)-polyacrylamide gel copolymerized with gelatin used for the second dimension. Proteases and protease inhibitors separated by IEF gel were applied on the second gel without trichloroacetic ...

  1. Direct cloning of specific genomic DNA sequences in plasmid libraries following fragment enrichment.

    OpenAIRE

    Nicholls, R D; Hill, A V; Clegg, J B; Higgs, D R

    1985-01-01

    We describe a simple method to directly clone any DNA fragment for which a flanking restriction enzyme map is known. Genomic DNA is digested with multiple enzymes cutting outside the fragment to be cloned, selected by electroelution from an agarose gel, and cloned directly into a plasmid vector. It is only necessary to screen 10-1000 colonies and recombinant DNA is ready for immediate molecular analysis without further subcloning. The use of this technique is demonstrated for the cloning of a...

  2. Histological and histochemical evaluation of human oral mucosa constructs developed by tissue engineering

    OpenAIRE

    Sánchez-Quevedo, M.C.; Alaminos, M.; Capitan, L.M.; Moreu, G.; Garzon, I.; Crespo, P.V.; Campos, A.

    2007-01-01

    Reconstruction of large oral mucosa defects is often challenging, since the shortage of healthy oral mucosa to replace the excised tissues is very common. In this context, tissue engineering techniques may provide a source of autologous tissues available for transplant in these patients. In this work, we developed a new model of artificial oral mucosa generated by tissue engineering using a fibrin-agarose scaffold. For that purpose, we generated primary cultures of human oral mucosa ...

  3. DNA-Based Genetic Markers for Rapid Cycling Brassica Rapa (Fast Plants Type) Designed for the Teaching Laboratory

    OpenAIRE

    Slankster, Eryn E.; Chase, Jillian M.; Jones, Lauren A.; Wendell, Douglas L.

    2012-01-01

    We have developed DNA-based genetic markers for rapid cycling Brassica rapa (RCBr), also known as Fast Plants. Although markers for B. rapa already exist, ours were intentionally designed for use in a teaching laboratory environment. The qualities we selected for were robust amplification in PCR, polymorphism in RCBr strains, and alleles that can be easily resolved in simple agarose slab gels. We have developed two single nucleotide polymorphism (SNP) based markers and 14 variable number tand...

  4. High-Throughput Plasmid Content Analysis of Borrelia burgdorferi B31 by Using Luminex Multiplex Technology▿ †

    OpenAIRE

    Norris, Steven J; Howell, Jerrilyn K.; Odeh, Evelyn A.; Lin, Tao; Gao, Lihui; Diane G Edmondson

    2010-01-01

    Borrelia burgdorferi, the causative agent of Lyme disease in North America, is an invasive pathogen that causes persistent multiorgan manifestations in humans and other mammals. Genetic studies of this bacterium are complicated by the presence of multiple plasmid replicons, many of which are readily lost during in vitro culture. The analysis of B. burgdorferi plasmid content by plasmid-specific PCR and agarose gel electrophoresis or other existing techniques is informative, but these techniqu...

  5. Serum low density lipoprotein of alcoholic patients is chemically modified in vivo and induces apolipoprotein E synthesis by macrophages.

    OpenAIRE

    Lin, R C; Dai, J; Lumeng, L; Zhang, M Y

    1995-01-01

    This work was carried out to investigate the effect of alcohol drinking on serum LDL. Agarose gel electrophoresis showed that LDL samples from alcoholic patients without serious liver disease were more negatively charged and moved faster toward the cathode than LDL from nondrinking control subjects. Rabbit antibodies raised by using keyhole limpet hemocyanin modified in vitro by 4-hydroxynonenal or by acetaldehyde as immunogens reacted more strongly with patients' LDL than with control LDL, i...

  6. Electrophoresis of proteins and DNA on horizontal sodium dodecyl sulfate polyacrylamide gels

    OpenAIRE

    Colombo Paolo; Cascone Eleonora; Bellavia Daniele; Duro Giovanni; Di Fiore Renata; Costa Maria A; Izzo Vincenzo; Gioviale Maria C; Barbieri Rainer

    2006-01-01

    Abstract An inexpensive Plexiglas apparatus which allows a simple and rapid preparation of horizontal polyacrylamide gels of different dimensions for different purposes, is described. Preparation of such gels is as easy and rapid as agarose gel preparation, and polymerized polyacrylamide gels are used to fractionate proteins or small DNA fragments using a common horizontal electrophoretic tank. This apparatus was used to electrophoretically fractionate proteins or DNA for immuno-blot analyses...

  7. Single bead-based electrochemical biosensor

    OpenAIRE

    LIU, CHANGCHUN; Schrlau, Michael G.; Bau, Haim H.

    2009-01-01

    A simple, robust, single bead-based electrochemical biosensor was fabricated and characterized. The sensor’s working electrode consists of an electrochemically-etched platinum wire, with a nominal diameter of 25 μm, hermetically heat-fusion sealed in a pulled glass capillary (micropipette). The sealing process does not require any epoxy or glue. A commercially available, densely functionalized agarose bead was mounted on the tip of the etched platinum wire. The use of a pre-functionalized bea...

  8. Telomere repeat DNA forms a large non-covalent complex with unique cohesive properties which is dissociated by Werner syndrome DNA helicase in the presence of replication protein A

    OpenAIRE

    Ohsugi, Itaru; Tokutake, Yoshiki; Suzuki, Noriyuki; IDE, TOSHINORI; Sugimoto, Masanobu; Furuichi, Yasuhiro

    2000-01-01

    We describe the unique structural features of a large telomere repeat DNA complex (TRDC) of >20 kb generated by a simple PCR using (TTAGGG)4 and (CCCTAA)4 as both primers and templates. Although large, as determined by conventional agarose gel electrophoresis, the TRDC was found to consist of short single-stranded DNA telomere repeat units of between several hundred and 3000 bases, indicating that it is a non-covalent complex comprising short cohesive telomere repeat units. S1 nuclease digest...

  9. Electroeluting DNA Fragments

    OpenAIRE

    Zarzosa-Álvarez, Ana L.; Sandoval-Cabrera, Antonio; Torres-Huerta, Ana L.; Ma. Bermudez-Cruz, Rosa

    2010-01-01

    Purified DNA fragments are used for different purposes in Molecular Biology and they can be prepared by several procedures. Most of them require a previous electrophoresis of the DNA fragments in order to separate the band of interest. Then, this band is excised out from an agarose or acrylamide gel and purified by using either: binding and elution from glass or silica particles, DEAE-cellulose membranes, "crush and soak method", electroelution or very often expensive commercial purification ...

  10. Synthesis of plus strands of retroviral DNA in cells infected with avian sarcoma virus and mouse mammary tumor virus.

    OpenAIRE

    Kung, H J; Fung, Y. K.; Majors, J E; Bishop, J M; Varmus, H E

    1981-01-01

    The vast majority of plus strands synthesized in quail cells acutely infected with avian sarcoma virus were subgenomic in size, generally less than 3 kilobases (kb). A series of discrete species could be identified after agarose gel electrophoresis by annealing with various complementary DNAs, indicating specificity in the initiation and termination of plus strands. The first plus strand to appear (within 2 h postinfection) was similar in length to the long redundancy at the ends of linear DN...

  11. Human mast cell chymase induces the accumulation of neutrophils, eosinophils and other inflammatory cells in vivo

    OpenAIRE

    He, Shaoheng; Walls, Andrew F.

    1998-01-01

    The roles of chymase in acute allergic responses are not clear, despite the relative abundance of this serine proteinase in the secretory granules of human mast cells. We have isolated chymase to high purity from human skin tissue by heparin-agarose affinity chromatography and Sephacryl S-200 gel filtration procedures, and have investigated the ability of human mast cell chymase to stimulate cell accumulation following injection into laboratory animals.Injection of chymase provoked marked neu...

  12. Controlling variation in the comet assay

    OpenAIRE

    Collins, Andrew R; El Yamani, Naouale; Lorenzo, Yolanda; Shaposhnikov, Sergey; Brunborg, Gunnar; Azqueta, Amaya

    2014-01-01

    Variability of the comet assay is a serious issue, whether it occurs from experiment to experiment in the same laboratory, or between different laboratories analysing identical samples. Do we have to live with high variability, just because the comet assay is a biological assay rather than analytical chemistry? Numerous attempts have been made to limit variability by standardizing the assay protocol, and the critical steps in the assay have been identified; agarose concentration, duration of ...

  13. Dynamic Loading of Deformable Porous Media Can Induce Active Solute Transport

    OpenAIRE

    Albro, Michael B.; Chahine, Nadeen O; Li, Roland; Yeager, Keith; Hung, Clark T.; Ateshian, Gerard A.

    2008-01-01

    Active solute transport mediated by molecular motors across porous membranes is a well-recognized mechanism for transport across the cell membrane. In contrast, active transport mediated by mechanical loading of porous media is a non-intuitive mechanism that has only been predicted recently from theory, but not yet observed experimentally. This study uses agarose hydrogel and dextran molecules as a model experimental system to explore this mechanism. Results show that dynamic loading can enha...

  14. Evaluation of electrophoretic profile and albumin quota in the cerebrospinal fluid of dogs with distemper showing or not neurvous signs Avaliação do perfil eletroforético e da cota de albumina do líquido cerebrospinal de cães acometidos pela cinomose apresentando ou não sinais neurológicos

    OpenAIRE

    F.G.V. Gama; C.T. Nishimori; M.R. Sobreira; Santana, A. E.

    2007-01-01

    The electrophoretic profile of cerebrospinal fluid proteins and albumin quota was studied in healthy dogs and dogs with distemper in either nervous or non-nervous phases. Cerebrospinal fluid (CSF) samples from 30 dogs were collected by puncture of the cisterna magna. The total protein content, the albumin quota, and the electrophoretic fraction of CSF proteins in agarose gel plates were evaluated. Results were similar in healthy dogs and dogs with distemper and no nervous signs, but were sign...

  15. Nano-Electrochemistry and Nano-Electrografting with an Original Combined AFM-SECM

    OpenAIRE

    Ammar Ben Brahim; Cédric Goyer; Christophe Demaille; Serge Palacin; Julienne Charlier; Federico Grisotto; Achraf Ghorbal

    2013-01-01

    This study demonstrates the advantages of the combination between atomic force microscopy and scanning electrochemical microscopy. The combined technique can perform nano-electrochemical measurements onto agarose surface and nano-electrografting of non-conducting polymers onto conducting surfaces. This work was achieved by manufacturing an original Atomic Force Microscopy-Scanning ElectroChemical Microscopy (AFM-SECM) electrode. The capabilities of the AFM-SECM-electrode were tested with the ...

  16. Serial thick, frozen, gallocyanin stained sections of human central nervous system

    OpenAIRE

    Heinsen, Helmut; Heinsen, Y. L.

    2010-01-01

    A rapid method for macroscopic and microscopic investigation of human CNS is proposed. After fonnalin fixation, gelatin or agarose embedding, and cryoprotective treatment, frozen human spinal cords, brainstems, or hemispheres can be serially cut into 0.7 mm thick slices. Stained with gallocyanin-chromalum, these slices facilitate cytoarchitectonic, neuropathologic, and quantitative examination. Regions of interest from parallel fonnalin-stored unstained slices can be embedded into paraffin an...

  17. Physical characterization of plasmids determining synthesis of a microcin which inhibits methionine synthesis in Escherichia coli.

    OpenAIRE

    Perez-Diaz, J C; Clowes, R C

    1980-01-01

    Plasmid deoxyribonucleic acid (DNA) isolated from each of three antibiotic-resistant clinical strains of Escherichia coli producing the same microcin showed multiple bands upon agarose gel electrophoresis. Transformants selected either for microcin resistance or ampicillin resistance yielded plasmid DNA corresponding in size to only one of the multiple bands. Plasmids, isolated from all three hosts, which determined microcin resistance and microcin production measured about 4 megadaltons by s...

  18. Enhanced xylitol production using immobilized Candida tropicalis with non-detoxified corn cob hemicellulosic hydrolysate

    OpenAIRE

    Yewale, Tatyaso; Panchwagh, Shruti; Rajagopalan, Srinivasan; Dhamole, Pradip B.; Jain, Rishi

    2016-01-01

    This study reports an industrially applicable non-sterile xylitol fermentation process to produce xylitol from a low-cost feedstock like corn cob hydrolysate as pentose source without any detoxification. Different immobilization matrices/mediums (alginate, polyvinyl alcohol, agarose gel, polyacrylamide, gelatin, and κ-carrageenan) were studied to immobilize Candida tropicalis NCIM 3123 cells for xylitol production. Amongst this calcium alginate, immobilized cells produced maximum amount of xy...

  19. Assessment of Growth Factor Treatment on Fibrochondrocyte and Chondrocyte Co-Cultures for TMJ Fibrocartilage Engineering

    OpenAIRE

    Kalpakci, Kerem N.; Kim, Eric J.; Athanasiou, Kyriacos A.

    2010-01-01

    Treatments for patients suffering from severe temporomandibular joint (TMJ) dysfunction are limited, motivating the development of strategies for tissue regeneration. In this study, co-cultures of fibrochondrocytes (FC) and articular chondrocytes (AC) were seeded in agarose wells, and supplemented with growth factors, to engineer tissue with biomechanical properties and ECM composition similar to native TMJ fibrocartilage. In the first phase, growth factors were applied alone and in combinati...

  20. Isolation and identification of a Giardia lamblia-specific stool antigen (GSA 65) useful in coprodiagnosis of giardiasis.

    OpenAIRE

    Rosoff, J D; Stibbs, H H

    1986-01-01

    A Giardia lamblia-specific antigen (GSA 65) was isolated from stools of G. lamblia-positive patients by crossed- and line-immunoelectrophoresis and counterimmunoelectrophoresis (CIE) in agarose by using rabbit antiserum prepared against G. lamblia cysts. CIE with rabbit anti-GSA 65 monospecific antiserum revealed that GSA 65 was present in aqueous stool eluates of giardiasis patients and in cysts and trophozoites of the parasite. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of im...

  1. Identification of pregnancy-associated glycoproteins and alpha-fetoprotein in fallow deer (Dama dama) placenta

    OpenAIRE

    Bériot, Mathilde; Tchimbou Njanjo, Aline Flora; Barbato, Olimpia; Beckers, Jean-François; Melo de Sousa, Noelita

    2014-01-01

    Background: This paper describes the isolation and characterization of pregnancy-associated glycoproteins (PAG) from fetal cotyledonary tissue (FCT) and maternal caruncular tissue (MCT) collected from fallow deer (Dama dama) pregnant females. Proteins issued from FCT and MCT were submitted to affinity chromatographies by using Vicia villosa agarose (VVA) or anti-bovine PAG-2 (R#438) coupled to Sepharose 4B gel. Finally, they were characterized by SDSPAGE and N-terminal microsequencing. Result...

  2. Development and mapping of SNP assays in allotetraploid cotton

    OpenAIRE

    Byers, Robert L.; Harker, David B.; Yourstone, Scott M.; Peter J Maughan; Udall, Joshua A.

    2012-01-01

    A narrow germplasm base and a complex allotetraploid genome have made the discovery of single nucleotide polymorphism (SNP) markers difficult in cotton (Gossypium hirsutum). To generate sequence for SNP discovery, we conducted a genome reduction experiment (EcoRI, BafI double digest, followed by adapter ligation, biotin–streptavidin purification, and agarose gel separation) on two accessions of G. hirsutum and two accessions of G. barbadense. From the genome reduction experiment, a total of 2...

  3. Gel versus capillary electrophoresis genotyping for categorizing treatment outcomes in two anti-malarial trials in Uganda

    OpenAIRE

    Hubbard Alan E; Dorsey Grant; Gupta Vinay; Rosenthal Philip J; Greenhouse Bryan

    2010-01-01

    Abstract Background Molecular genotyping is performed in anti-malarial trials to determine whether recurrent parasitaemia after therapy represents a recrudescence (treatment failure) or new infection. The use of capillary instead of agarose gel electrophoresis for genotyping offers technical advantages, but it is unclear whether capillary electrophoresis will result in improved classification of anti-malarial treatment outcomes. Methods Samples were genotyped using both gel and capillary elec...

  4. New variants of alpha 1-antitrypsin: comparison of Pi typing techniques.

    OpenAIRE

    Cox, D. W.

    1981-01-01

    Four new rare inherited variants (Pi types) of alpha 1-antitrypsin (alpha 1-protease inhibitor) are described. Each variant has been compared with previously reported genetic variants by several techniques used for Pi typing: isoelectric focusing in polyacrylamide gel, starch gel electrophoresis, and agarose gel electrophoresis. Some variants are identical or very similar by one technique but can be clearly distinguished by another technique. Crossed immunoelectrophoresis and gel immunofixati...

  5. Determination of microbial genome sizes by two-dimensional denaturing gradient gel electrophoresis.

    OpenAIRE

    Poddar, S K; Maniloff, J

    1989-01-01

    In two-dimensional denaturing gradient gel electrophoresis, DNA is digested with a restriction endonuclease and the resulting DNA fragments are separated as a function of size by conventional agarose gel electrophoresis. Following this first dimension electrophoresis, the fragment distribution is placed at the top of a denaturing gradient slab gel and electrophoresis is carried out parallel to the gradient direction. This second dimension separation is a complex function of the base sequence ...

  6. Small angle neutron scattering from DNA molecules during gel electrophoresis

    International Nuclear Information System (INIS)

    We have performed small angle neutron scattering experiments on agarose-DNA gels undergoing electrophoresis. Two kinds of DNA (5 and 50 kilobase pairs) were used with applied fields up to 5 V/cm. The SANS patterns obtained do not show evidence of any anisotropic scattering. This result is discussed in the context of current theories of DNA fragments migrating through a polysaccharide network. (orig.)

  7. Thermally forced transitions of DNA-CTMA complex microstructure

    Science.gov (United States)

    Nizioł, Jacek; Ekiert, Robert; Śniechowski, Maciej; Słomiany, Magdalena; Marzec, Mateusz M.

    2016-06-01

    DNA complexed with amphiphilic cationic surfactants is a new class of optical material. In this work DNA and its complex with cetyltrimetyl ammonium chloride were thermally annealed. X-ray diffractometry revealed irreversible changes of DNA-CTMA microstructure. The new microstucture that appeared in result of the first heating course was stable, despite the further thermal annealing. Agarose gel electrophoresis indicated fundamental differences between thermally treated native DNA and DNA-CTMA complex.

  8. Protocol: a rapid and economical procedure for purification of plasmid or plant DNA with diverse applications in plant biology

    OpenAIRE

    Li Li; Li Jian-Feng; Sheen Jen

    2010-01-01

    Abstract Research in plant molecular biology involves DNA purification on a daily basis. Although different commercial kits enable convenient extraction of high-quality DNA from E. coli cells, PCR and agarose gel samples as well as plant tissues, each kit is designed for a particular type of DNA extraction work, and the cost of purchasing these kits over a long run can be considerable. Furthermore, a simple method for the isolation of binary plasmid from Agrobacterium tumefaciens cells with s...

  9. Use of Multienzyme Multiplex PCR Amplified Fragment Length Polymorphism Typing in Analysis of Outbreaks of Multiresistant Klebsiella pneumoniae in an Intensive Care Unit

    OpenAIRE

    van der Zee, Anneke; Steer, Niels; Thijssen, Eveline; Nelson, Jolande; van't Veen, Annemarie; Buiting, Anton

    2003-01-01

    We developed and optimized a new modified amplified fragment length polymorphism (AFLP) typing method to obtain a multibanding fingerprint that can be separated by agarose gel electrophoresis. Both to maximize the discriminatory power and to facilitate the computer-assisted analysis, bacterial DNA was digested with four different restriction enzymes. After ligation of adaptors to the DNA fragments, PCR testing of various single primers was performed. Two single primers that gave optimal resul...

  10. Phos-tag beads as an immunoblotting enhancer for selective detection of phosphoproteins in cell lysates

    OpenAIRE

    Kinoshita, Eiji; Kinoshita-Kikuta, Emiko; Koike, Tohru

    2009-01-01

    The low specificity of anti-phosphoprotein antibodies is often a problem in immunoblotting analyses. We introduce a simple pretreatment procedure for cell lysates to give more-specific detection of phosphoproteins in immunoblotting. Cellular phosphoproteins were preferentially trapped on Phos-tag agarose phosphate-affinity beads in a homemade spin-centrifuge microtube unit, and nonphosphorylated proteins were excluded in the filtrate. The phosphoprotein-bound beads suspended in a sample-loadi...

  11. Lateral View Flow System for Studies of Cell Adhesion and Deformation under Flow Conditions

    OpenAIRE

    Yuan, Jin; Melder, Robert J; Jain, Rakesh K.; Munn, Lance L.

    2001-01-01

    Physical interactions between circulating cells and the vascular wall play a central role in inflammation, metastasis, atherosclerosis, and therapeutic cell delivery. Unfortunately, traditional in vitro flow assays cannot be used to visualize the details of cell-surface interactions in blood flow because of inappropriate geometry and the poor penetration of light in erythrocyte solutions. To overcome these obstacles, we have developed an agarose-cast cylindrical vessel system to examine the p...

  12. Validation of a Clostridium Endospore Viability Assay and Analysis of Greenland Ices and Atacama Desert Soils▿ †

    OpenAIRE

    Yang, Wan-Wan; Ponce, Adrian

    2011-01-01

    A microscopy-based endospore viability assay (micro-EVA) capable of enumerating germinable Clostridium endospores (GCEs) in less than 30 min has been validated and employed to determine GCE concentrations in Greenland ices and Atacama Desert soils. Inoculation onto agarose doped with Tb3+ and d-alanine triggers Clostridium spore germination and the concomitant release of ∼108 molecules of dipicolinic acid (DPA) per endospore, which, under pulsed UV excitation, enables enumeration of resultant...

  13. Serological analysis of human IgG and IgE anti-insulin antibodies by solid-phase radioimmunoassays

    International Nuclear Information System (INIS)

    A single solid-phase assay system which is useful for quantitative measurement of both IgG and IgE anti-insulin antibodies in human serum has been developed. Insulin-specific immunoglobulins are absorbed from human serum by excess quantities of insulin-agarose. After washes to remove unbound immunoglobulins, radioiodinated Staph A or rabbit anti-human IgE is added to detect bound IgG or IgE anbitodies, respectively

  14. Modified paired end rapid library preparation protocol for 454 GS Junior 8 kb library preparation using Covaris g-tubes and BluePippin electrophoresis

    OpenAIRE

    sprotocols

    2014-01-01

    Authors: Janet Hill, Bonnie Chaban, Jennifer Town, Matthew Links & Tim Dumonceaux ### Abstract This protocol describes an alternative approach to performing Roche’s Paired End Rapid Library Preparation Method for 8 kb span libraries. This method uses the Corvaris g-tube for DNA fragmentation, eliminating the need for a HydroShear apparatus, and a Sage Science BluePippin electrophoresis platform to size select the 8 kb fragments, eliminating the need for agarose gel electrophoresi...

  15. Serum proteins analysis by capillary electrophoresis

    OpenAIRE

    Uji, Yoshinori; Okabe, Hiroaki

    2001-01-01

    The purpose of this study was to evaluate the efficacy of multi-capillary electrophoresis instrument in clinical laboratory. An automated clinical capillary electrophoresis system was evaluated for performing serum proteins electrophoresis and immuno-fixation electrophoresis by subtraction. In this study the performance of capillary electrophoresis was compared with the cellulose acetate membrane electrophoresis and agarose gel immunofixation electrophoresis for serum proteins. The results of...

  16. Human Immunodeficiency Virus Type 1 DNA Sequences Genetically Damaged by Hypermutation Are Often Abundant in Patient Peripheral Blood Mononuclear Cells and May Be Generated during Near-Simultaneous Infection and Activation of CD4+ T Cells

    OpenAIRE

    Janini, Mario; Rogers, Melissa; Birx, Deborah R.; McCutchan, Francine E.

    2001-01-01

    G-to-A hypermutation has been sporadically observed in human immunodeficiency virus type 1 (HIV-1) proviral sequences from patient peripheral blood mononuclear cells (PBMC) and virus cultures but has not been systematically evaluated. PCR primers matched to normal and hypermutated sequences were used in conjunction with an agarose gel electrophoresis system incorporating an AT-binding dye to visualize, separate, clone, and sequence hypermutated and normal sequences in the 297-bp HIV-1 proteas...

  17. Cellular differentiation in 3D-bioprinted mesenchymal stem cell-loaded hydrogels with varying structural and mechanical properties

    OpenAIRE

    Duarte Campos, Daniela Filipa

    2016-01-01

    Hydrogels are a promising alternative to rigid biomaterials typically used in the field of bone tissue engineering for the treatment of musculoskeletal disorders. By hydrogel-based 3D-bioprinting, the native ornamentation of cells and matrix from bone tissue could be resembled. Herein, it was hypothesized the combination of polysaccharides (agarose, alginate) with biological components (collagen, fibrinogen) would increase mechanical stiffness of printed constructs as well as support the prin...

  18. Glutathione S-transferase activity and isoenzyme composition in benign ovarian tumours, untreated malignant ovarian tumours, and malignant ovarian tumours after platinum/cyclophosphamide chemotherapy.

    OpenAIRE

    Zee, A.G. Van der; van Ommen, B.; Meijer, C; Hollema, H; Bladeren, P.J. van; de Vries, E. G.

    1992-01-01

    Glutathione S-transferase (GST) isoenzyme composition, isoenzyme quantities and enzymatic activity were investigated in benign (n = 4) ovarian tumours and malignant ovarian tumours, before (n = 20) and after (n = 16) chemotherapy. Enzymatic activity of GST in cytosols was measured by determining 1-chloro-2,4-dinitrobenzene conjugation with glutathione, cytosolic GST subunits were determined by wide pore reversed phase HPLC, using a S-hexylglutathione-agarose affinity column, and isoelectric f...

  19. Prominent mitochondrial DNA recombination intermediates in human heart muscle

    OpenAIRE

    Kajander, Olli A; Karhunen, Pekka J.; Holt, Ian J.; Jacobs, Howard T.

    2001-01-01

    Recombination intermediates containing four-way (Holliday) junctions are generated during DNA repair and replication in many systems, including yeast mitochondrial DNA (mtDNA). In contrast, convincing evidence for recombination in mammalian mtDNA is lacking. We have used two-dimensional agarose-gel electrophoresis to analyse non-linear forms of mtDNA in human heart muscle. Replication intermediates from both the coupled and strand-asynchronous mtDNA replication pathways were detected. An addi...

  20. Investigation of an outbreak of antibiotic-associated colitis by various typing methods.

    OpenAIRE

    Wüst, J; Sullivan, N M; Hardegger, U; Wilkins, T D

    1982-01-01

    During an outbreak of diarrheal disease due to Clostridium difficile in a surgical ward, 16 C. difficile isolates were cultured from fecal samples of 15 patients. Agarose gel electrophoresis for the detection of plasmid DNA, crossed immunoelectrophoresis for the detection of extracellular antigens and toxins, polyacrylamide gel electrophoresis for analyses of soluble proteins, assays for cytotoxicity, and a comparison of susceptibility to antimicrobial agents were employed. At least 12 of the...

  1. Longevity is independent of common variations in genes associated with cardiovascular risk

    DEFF Research Database (Denmark)

    Bladbjerg, E M; Andersen-Ranberg, K; Maat, M de; Kristensen, S R; Jeune, B; Gram, J; Jespersen, J

    1999-01-01

    -specific amplification followed by agarose gel electrophoresis. The frequencies of the high-risk alleles in centenarians were: for FVII R/Q353 0.91; for FVII intron 7 (37bp)n 0.67; for FVII-323 ins10 0.90; for fibrinogen 0.16; for PAI-1 0.52; for t-PA 0.59; for GPIIb/IIIa 0.16; for prothrombin 0.008; for MTHFR 0.33; for...

  2. Two dimensional (2-D) electrophoresis and silver staining of proteins in SDS gels

    International Nuclear Information System (INIS)

    Two dimensional electrophoresis is a powerful way to analyze protein samples. It separates molecules by charge, then by size in a polyacrylamide or agarose gel. Combining these two techniques allows visualization of up to 1000 peptides on a single gel. Silver staining of proteins provides a rapid and ultra-sensitive method for the detection of nanogram amounts of proteins. The procedures for two dimensional electrophoresis and silver staining of proteins in gels are described

  3. Estimation of circular DNA size using gamma-irradiation and pulsed-field gel electrophoresis

    International Nuclear Information System (INIS)

    A method is described for estimating the size of large circular DNAs found within complex chromosomal DNA preparations. DNAs are treated with low levels of gamma-irradiation, sufficient to introduce a single double-stranded break per circle, and the resulting linear DNA is sized by pulsed-field electrophoresis and blot hybridization. The method is fast, reproducible, and very conveniently applied to the agarose-enclosed chromosomal DNA preparations commonly used in pulsed field electrophoresis

  4. Evaluation of eight and a half years of neonatal screening for haemoglobinopathies in Birmingham

    OpenAIRE

    Griffiths, Paul D; Mann, Jillian R; Darbyshire, Philip J; Green, Anne

    1988-01-01

    A pilot neonatal screening programme for haemoglobinopathies linked with screening for phenylketonuria and congenital hypothyroidism was reviewed. During 1978 to December 1986 137 000 neonates were tested. There were improvements in the detection rate and accuracy of diagnosis for homozygotes and mixed heterozygotes, mainly associated with the introduction of citrate agarose gel electrophoresis as a follow up procedure on all specimens showing any abnormality on the initial cellulose acetate ...

  5. Rapid method for epidemiological evaluation of gram-positive cocci by field inversion gel electrophoresis.

    OpenAIRE

    Goering, R V; Winters, M A

    1992-01-01

    We report a rapid method for the isolation of intact chromosomal DNA from gram-positive cocci that is suitable for in situ restriction endonuclease digestion in agarose blocks. When combined with a rapid field inversion gel electrophoresis protocol, this approach allows the preparation and electrophoretic analysis of chromosomal restriction fragments produced by rare-cutting enzymes in a total time period of 2 days from start to finish. The utility of the method is demonstrated in the epidemi...

  6. Mapping genomic organization by field inversion and two-dimensional gel electrophoresis: application to the murine T-cell receptor gamma gene family

    OpenAIRE

    Woolf, T; Lai, E; Kronenberg, M; Hood, L

    1988-01-01

    A new two-dimensional gel electrophoresis technique has been developed for the mapping of multigene families. Resolution in the first dimension is based on the generation of large size DNA fragments by infrequently-cutting restriction enzymes, and separation of these fragments by field inversion gel (FIG) electrophoresis. A second restriction enzyme digestion is then carried out with the separated DNA fragments in the agarose gel. Standard gel electrophoresis in the second dimension allows on...

  7. Rapid Diagnosis of Herpes Simplex Virus Infection by a Loop-Mediated Isothermal Amplification Method

    OpenAIRE

    Enomoto, Yoshihiko; Yoshikawa, Tetsushi; Ihira, Masaru; Akimoto, Shiho; Miyake, Fumi; Usui, Chie; Suga, Sadao; Suzuki, Kayoko; Kawana, Takashi; Nishiyama, Yukihiro; Asano, Yoshizo

    2005-01-01

    Primers for herpes simplex virus type 1 (HSV 1)-specific loop-mediated isothermal amplification (LAMP) method amplified HSV-1 DNA, while HSV-2-specific primers amplified only HSV-2 DNA; no LAMP products were produced by reactions performed with other viral DNAs. The sensitivities of the HSV-1- and HSV-2-specific LAMP methods, determined by agarose gel electrophoresis, reached 500 and 1,000 copies/tube, respectively. The turbidity assay, however, determined the sensitivity of the HSV-1- and HS...

  8. Lectinlike adhesins in the Bacteroides fragilis group.

    OpenAIRE

    Rogemond, V; Guinet, R M

    1986-01-01

    Lectinlike adhesins were identified in the Bacteroides fragilis group by using sugars immobilized on agarose beads either with whole bacteria by direct microscopic examination or with soluble extracts by immunoaffinoelectrophoresis. These two methods allowed the identification of two sugars reacting with whole bacteria and with the corresponding extracts: alpha-D-glucosamine and D-galactosamine. Among eight strains tested representing seven species, the two strains of B. fragilis were equally...

  9. Incomplete primer extension during in vitro DNA amplification catalyzed by Taq polymerase; exploitation for DNA sequencing.

    OpenAIRE

    Olsen, D. B.; Eckstein, F.

    1989-01-01

    Polyacrylamide gel electrophoresis of DNA fragments obtained by the polymerase chain reaction using Taq polymerase revealed the presence of multiple fragments shorter than the expected product. These abortive extension products were observed even when analysis by agarose gel electrophoresis showed only a single band. The production of prematurely terminated fragments can be exploited for the sequencing of PCR products if phosphorothioate groups are incorporated base specifically during the re...

  10. A Molecular Fraction Collecting Tool for the ABI 310 Automated Sequencer

    OpenAIRE

    Lin, Ming-Tseh; Rich, Roy G.; Shipley, Royce F.; Hafez, Michael J.; Tseng, Li-Hui; Murphy, Kathleen M.; Gocke, Christopher D.; Eshleman, James R.

    2007-01-01

    Several methods exist to retrieve and purify DNA fragments after agarose or polyacrylamide gel electrophoresis for subsequent analyses. However, molecules present in low concentration and molecules similar in size to their neighbors are difficult to purify. Capillary electrophoresis has become popular in molecular diagnostic laboratories because of its automation, excellent resolution, and high sensitivity. In the current study, the ABI Prism 310 Genetic Analyzer was reconfigured into a fract...

  11. Genome analysis of Bradyrhizobium japonicum serocluster 123 field isolates by using field inversion gel electrophoresis.

    OpenAIRE

    Sobral, B W; Sadowsky, M. J.; Atherly, A G

    1990-01-01

    The genomes of 11 Bradyrhizobium japonicum serocluster 123 field isolates were analyzed by using field inversion gel electrophoresis. Genomic fingerprints produced by digestion of intact genomic DNA in agarose plugs with the rare-cutting restriction enzymes AseI, DraI, SpeI, and XbaI showed that the isolates were genetically diverse. Few (30 to 50%) isolates exhibited the same fingerprint as the USDA serogroup strain with which they are antigenically related. Southern hybridization with a nif...

  12. Scrambling of bands in gel electrophoresis of DNA.

    OpenAIRE

    Lalande, M; Noolandi, J; Turmel, C; Brousseau, R.; Rousseau, J.; Slater, G W

    1988-01-01

    Under certain conditions of agarose gel electrophoresis, larger DNA molecules migrate faster than smaller ones. This anomalous mobility of DNA, which can lead to serious errors in the measurement of DNA fragment lengths, is related to near-zero velocity conformations which can trap DNA chains during electrophoresis. Intermittent electric fields can be used to alter the chain conformations so as to restore the monotonic mobility-size relationship which is necessary for a correct interpretation...

  13. Assessment of clone identity and sequence fidelity for 1189 IMAGE cDNA clones

    OpenAIRE

    Halgren, Robert G.; Fielden, Mark R.; Fong, Cora J.; Zacharewski, Timothy R

    2001-01-01

    This report documents the error rate in a commercially distributed subset of the IMAGE Consortium mouse cDNA clone collection. After isolation of plasmid DNA from 1189 bacterial stock cultures, only 62.2% were uncontaminated and contained cDNA inserts that had significant sequence identity to published data for the ordered clones. An agarose gel electrophoresis pre-screening strategy identified 361 stock cultures that appeared to contain two or more plasmid species. Is...

  14. Pulsed-field gel electrophoretic analysis of leptospiral DNA.

    OpenAIRE

    Taylor, K A; Barbour, A. G; Thomas, D D

    1991-01-01

    The genomic structures of spirochete species are not well characterized, and genetic studies on these organisms have been hampered by lack of a genetic exchange mechanism in these bacteria. In view of these observations, pulsed-field gel electrophoresis was used to examine the genomes of Leptospira species. Live cells, prepared in agarose plugs, were lysed in situ, and the DNA was analyzed under different electrophoretic conditions. Pulsed-field gel electrophoresis of DNA digested with infreq...

  15. Parameters of field inversion gel electrophoresis for the analysis of pox virus genomes.

    OpenAIRE

    Bostock, C J

    1988-01-01

    The effects of variation in the lengths of forward and reverse pulses, voltage gradient, gel concentration and gel temperature on the mobility of DNA molecules in agarose gels during field inversion gel electrophoresis (FIGE) have been determined. A curve, which best fits the empirical data, is presented and allows the choice of pulse conditions and voltage gradient most suitable for the resolution of molecules of chosen size. The use of FIGE in the analysis and direct mapping of large virus ...

  16. Novel display of knotted DNA molecules by two-dimensional gel electrophoresis

    OpenAIRE

    Trigueros, Sonia; Arsuaga, Javier; Vázquez, María E.; Sumners, De Witt; Roca, Joaquim

    2001-01-01

    We describe a two-dimensional agarose gel electrophoresis procedure that improves the resolution of knotted DNA molecules. The first gel dimension is run at low voltage, and DNA knots migrate according to their compactness. The second gel dimension is run at high voltage, and DNA knots migrate according to other physical parameters such as shape and flexibility. In comparison with one-dimensional gel electrophoresis, this procedure segregates the knotted DNA molecules ...

  17. Capillary electrophoresis as a technique to analyze sequence-induced anomalously migrating DNA fragments.

    OpenAIRE

    Wenz, H M

    1994-01-01

    Sequence-induced anomalous migration of double-stranded (ds) DNA in native gel electrophoresis is a well known phenomenon. The retardation of migration is more obvious in polyacrylamide compared with agarose gels, and is greatly affected by the concentration of the gel and the temperature. This anomalous migration results in a difference between calculated and actual sizes of the affected DNA fragments. A low viscosity polymer solution (DNA Fragment Analysis Reagent) under investigation for u...

  18. Intensive Linkage Mapping in a Wasp (Bracon Hebetor) and a Mosquito (Aedes Aegypti) with Single-Strand Conformation Polymorphism Analysis of Random Amplified Polymorphic DNA Markers

    OpenAIRE

    Antolin, M F; Bosio, C. F.; COTTON, J; W. Sweeney; Strand, M.R.; Black-IV, W. C.

    1996-01-01

    The use of random amplified polymorphic DNA from the polymerase chain reaction (RAPD-PCR) allows efficient construction of saturated linkage maps. However, when analyzed by agarose gel electrophoresis, most RAPD-PCR markers segregate as dominant alleles, reducing the amount of linkage information obtained. We describe the use of single strand conformation polymorphism (SSCP) analysis of RAPD markers to generate linkage maps in a haplodiploid parasitic wasp Bracon (Habrobracon) hebetor and a d...

  19. Alkaline gel electrophoresis assay to detect DNA strand breaks and repair mechanisms in Escherichia coli

    OpenAIRE

    José Carlos Pelielo De Mattos; Ellen Serri da Motta; Márcia Betania Nunes de Oliveira; Flávio José da Silva Dantas; Adriano Caldeira de Araujo

    2008-01-01

    Reactive oxygen species (ROS) can induce lesions in different cellular targets, including DNA. Stannous chloride (SnCl2) is a ROS generator, leading to lethality in Escherichia coli (E. coli), with the base excision repair (BER) mechanism playing a role in this process. Many techniques have been developed to detect genotoxicity, as comet assay, in eukaryotic cells, and plasmid DNA agarose gel electrophoresis. In this study, an adaptation of the alkaline gel electrophoresis method was carried ...

  20. Resolution of DNA molecules greater than 5 megabases by contour-clamped homogeneous electric fields.

    OpenAIRE

    Vollrath, D; Davis, R.W.

    1987-01-01

    Excellent resolution of chromosomal DNA molecules from Saccharomyces cerevisiae, Candida albicans and Schizosaccharomyces pombe has been obtained using alternating contour-clamped homogeneous electric field (CHEF) gel electrophoresis. The largest of these molecules is greater than 5 Mb in size and is resolved after 130 hours in a 0.6% agarose gel at a field strength of 1.3 V/cm and a switching interval of 1 hour. Separation of concatamers of phage lambda DNA reveals four regions of resolution...

  1. Cell-free translation of murine coronavirus RNA.

    OpenAIRE

    Leibowitz, J L; Weiss, S.R.; Paavola, E; Bond, C W

    1982-01-01

    The coding assignments of the intracellular murine hepatitis virus-specific subgenomic RNA species and murine hepatitis virion RNA have been investigated by cell-free translation. The six murine hepatitis virus-specific subgenomic RNAs were partially purified by agarose gel electrophoresis and translated in an mRNA-dependent rabbit reticulocyte lysate, and the cell-free translation products were characterized by gel electrophoresis, immunoprecipitation, and tryptic peptide mapping. These stud...

  2. A systematic study of field inversion gel electrophoresis.

    OpenAIRE

    Heller, C.; Pohl, F M

    1989-01-01

    The mobilities of oligomers of phage lambda DNA and of yeast chromosomes in agarose gels during field inversion gel electrophoresis (FIGE) were measured at different pulse times and electric fields. Also the ratios between forward and backward pulse times and/or field gradients were varied. The problem of 'band inversion' during FIGE, leading to an ambiguity in the mobility of large DNA fragments, was solved by using two dimensional gel electrophoresis with different parameters in the first a...

  3. Separations of open-circular DNA using pulsed-field electrophoresis.

    OpenAIRE

    Levene, S D; Zimm, B H

    1987-01-01

    The effect of high electric fields on the gel-electrophoretic mobility of open-circular DNA in agarose differs dramatically from that on linear molecules of the same molecular weight. At high fields, sufficiently large circular forms are prevented from migrating into the gel whereas linear molecules and smaller circular DNAs migrate normally. This effect is strongly field dependent, affecting circular molecules of decreasing size with increasing field strength. We have studied this effect wit...

  4. Detection of Mycobacterium tuberculosis in clinical samples by two-step polymerase chain reaction and nonisotopic hybridization methods.

    OpenAIRE

    Shawar, R M; el-Zaatari, F A; Nataraj, A; Clarridge, J E

    1993-01-01

    Detection of Mycobacterium tuberculosis in clinical specimens by the polymerase chain reaction (PCR) was compared with detection by culture. A 317-bp segment within the M. tuberculosis-specific insertion sequence IS6110 was amplified. The detection limit of the PCR assay for cultured mycobacteria was 50 cells per reaction by ethidium bromide-stained agarose gel electrophoresis and 5 cells per reaction by hybridization with an oligonucleotide probe conjugated with either digoxigenin or alkalin...

  5. Characterization of Proteinuria in Dogue de Bordeaux Dogs, a Breed Predisposed to a Familial Glomerulonephropathy: A Retrospective Study

    OpenAIRE

    Trumel, Catherine; Smets, Pascale M. Y.; Braun, Jean-Pierre; Aresu, Luca; Daminet, Sylvie; Concordet, Didier; Palanche, Florence; Peeters, Dominique

    2015-01-01

    Dogue de Bordeaux dog has been reported to be predisposed to a familial glomerulonephropathy that displays some morphological modifications reported in focal and segmental glomerulosclerosis. Prevalence of quantitatively abnormal renal proteinuria was recently reported to be 33% in this breed. The nature of the proteinuria was assessed by sodium dodecyl sulfate-agarose gel electrophoresis and determinations of urinary markers (urinary retinol-binding protein, urinary N-acetyl-beta-glucosamini...

  6. Plasmids and Protein Patterns of Escherichia coli Isolated from Bovine Mastitis in Konya, Turkey

    OpenAIRE

    UÇAN, Uçkun S.

    2005-01-01

    In this study, a total of 30 Escherichia coli isolates obtained from milk samples of dairy cows suffering from subclinical mastitis in Konya, Turkey were typed according to plasmids and protein patterns. Agarose gel electrophoresis and sodium doedecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) methods were used to identify plasmids and whole-cell protein profiles. Of these two methods, SDS-PAGE typing proved to be more discriminate for typing the isolates.

  7. Functional and Physical Outcomes following Use of a Flexible CO2 Laser Fiber and Bipolar Electrocautery in Close Proximity to the Rat Sciatic Nerve with Correlation to an In Vitro Thermal Profile Model

    OpenAIRE

    Robinson, A. M.; Fishman, A. J.; Bendok, B. R.; C.-P. Richter

    2015-01-01

    This study compared functional and physical collateral damage to a nerve when operating a Codman MALIS Bipolar Electrosurgical System CMC-III or a CO2 laser coupled to a laser, with correlation to an in vitro model of heating profiles created by the devices in thermochromic ink agarose. Functional damage of the rat sciatic nerve after operating the MALIS or CO2 laser at various power settings and proximities to the nerve was measured by electrically evoked nerve action potentials, and histolo...

  8. THE DEVELOPMENT OF BIOCHEMICAL OXYGEN DEMAND SENSOR USING LOCAL YEAST: Candida fukuyamaensis, UICC Y-247

    OpenAIRE

    Endang Saepudin; Fenny Triana Zulfia; Ivandini Tribidasari Anggraningrum

    2011-01-01

    In order to shorten the measurement time of biochemical oxygen demand (BOD), a BOD sensor based on yeastmetabolism was developed. Local yeast, Indonesian Origin, Candida fukuyamaensis UICC Y-247, was used as atransducer. The yeast was immobilized as a thin film in agarose matrix with the auxiliary of Nafion® acting as themembrane for ion exchange process. The film was then attached to gold-modified glassy carbons and used as transduceron the working electrodes. The measurements were conducted...

  9. Different sterilization methods for overcoming internal bacterial infection in sunflower seeds

    Directory of Open Access Journals (Sweden)

    Taški-Ajduković Ksenija J.

    2005-01-01

    Full Text Available During culture of protoplasts in agarose droplets, permanent problem was bacterial infection. It was assumed that the seeds are the origin of infection, so different sterilization methods were tested in order to overcome this problem. Germination, infection of seeds and hypocotyls and their growth were examined. Based on these parameters, the best result was obtained with the combined use of 5% commercial bleach and dry heating at 45°C.

  10. Effects of light intensities and photoperiods on growth and proteolytic activity in purple non-sulfur marine bacterium, Afifella marina strain ME (KC205142)

    OpenAIRE

    Sujjat Al-Azad; Tan Kar Soon; Julian Ransangan

    2013-01-01

    Afifella marina strain ME (KC205142), a purple non-sulfur bacterium was isolated from mangrove habitats of Sabah. The effects of light intensities and photoperiods on proteolytic activity in Afifella marina strain ME (KC205142) were investigated. Secretion of proteolytic enzymes in Afifella marina was preliminarily assessed by skim milk agarose media. Subsequently, light intensities, such as, dark, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500 and 5000 lux were used to ev...

  11. Anti-HIV antibodies in the CSF of AIDS patients: a serological and immunoblotting study.

    OpenAIRE

    Bukasa, K S; Sindic, Christian; Bodéus, Monique; Burtonboy, Guy; Laterre, C; Sonnet, J.

    1988-01-01

    CSF and serum samples from 16 AIDS patients were tested for the presence of anti-HIV antibodies either by classical serological methods or by an immunoblot technique based on agarose gel isoelectric focusing and transfer of the specific IgG antibodies onto HIV antigens-loaded nitrocellulose sheets. This method enabled the demonstration of an intrathecal synthesis of anti-HIV oligoclonal IgG antibodies, often superimposed on diffuse polyclonal production, in 14 patients. The two negative cases...

  12. Large Scale Library Generation for High Throughput Sequencing

    OpenAIRE

    Borgström, Erik; Lundin, Sverker; Lundeberg, Joakim

    2011-01-01

    Background Large efforts have recently been made to automate the sample preparation protocols for massively parallel sequencing in order to match the increasing instrument throughput. Still, the size selection through agarose gel electrophoresis separation is a labor-intensive bottleneck of these protocols. Methodology/Principal Findings In this study a method for automatic library preparation and size selection on a liquid handling robot is presented. The method utilizes selective precipitat...

  13. Design and Validation of Medical Devices for Photothermally Augmented Treatments

    OpenAIRE

    Andriani, Rudy Thomas

    2014-01-01

    *1-Dimensional Advective-Diffusion Model in Porous Media Infusion of therapeutic agents into tissue is makes use of two mass transport modes: advective transport, and molecular diffusion. Bulk infusion into a 0.6% wt agarose phantom was modeled as an infinite, homogenous, and isotropic porous medium saturated with the same solvent used in the infused dye tracer. The source is assumed to be spherical and isotropic with constant flow rate and concentration. The Peclet numberdecreases wit...

  14. Pesticide exposure and risk of monoclonal gammopathy of undetermined significance in the Agricultural Health Study

    OpenAIRE

    Landgren, Ola; Kyle, Robert A.; Hoppin, Jane A.; Beane Freeman, Laura E.; Cerhan, James R.; Katzmann, Jerry A.; Rajkumar, S. Vincent; Alavanja, Michael C

    2009-01-01

    Pesticides are associated with excess risk of multiple myeloma, albeit inconclusively. We included 678 men (30-94 years) from a well-characterized prospective cohort of restricted-use pesticide applicators to assess the risk of monoclonal gammopathy of undetermined significance (MGUS). Serum samples from all subjects were analyzed by electrophoresis performed on agarose gel; samples with a discrete or localized band were subjected to immunofixation. Age-adjusted prevalence estimates of MGUS w...

  15. Characterization of a satellite RNA associated with strain K8 of cucumber mosaic virus.

    OpenAIRE

    Fraile, Aurora; Moriones, E.; Garcia-Arenal, Fernando

    1990-01-01

    Cucumber mosaic virus strain K8 (K8-CMV), originally isolated in NW Italy from Cucumis metuliferus, was sent to us by Dr. Lisa after being passaged and multiplied in tobacco. Northern blots to probes against Fny-CMV (subgroup I) and Ls-CMV (subgroup II) showed K8-CMV to belong to subgroup I of CMV isolates (1). In agarose gel electrophoresis of virion encapsidated RNAs a fifth RNA was found, shown to be a satellite RNA (K8-sat RNA).

  16. Chromosome Polymorphisms among Strains of Hansenula polymorpha (syn. Pichia angusta)

    OpenAIRE

    Marri, Laura; Rossolini, Gian Maria; Satta, Giuseppe

    1993-01-01

    Contour-clamped homogeneous electrophoresis and an embedded-agarose method of sample preparation were combined to carry out an analysis of the chromosome sets of nine strains of Hansenula polymorpha (syn. Pichia angusta). Chromosomal DNA molecules could be separated into a series of bands ranging, approximately, from 650 up to 2,200 kb in size. Polymorphism of the electrophoretic pattern was demonstrated among the strains investigated in this study. Cross-hybridization between H. polymorpha a...

  17. Molecular Map of the Chlamydomonas reinhardtii Nuclear Genome

    OpenAIRE

    Kathir, Pushpa; LaVoie, Matthew; Brazelton, William J.; Haas, Nancy A.; Lefebvre, Paul A.; Silflow, Carolyn D.

    2003-01-01

    We have prepared a molecular map of the Chlamydomonas reinhardtii genome anchored to the genetic map. The map consists of 264 markers, including sequence-tagged sites (STS), scored by use of PCR and agarose gel electrophoresis, and restriction fragment length polymorphism markers, scored by use of Southern blot hybridization. All molecular markers tested map to one of the 17 known linkage groups of C. reinhardtii. The map covers approximately 1,000 centimorgans (cM). Any position on the C. re...

  18. The Cloning of the Human Tumor Supressor Gene INGI: DNA Cloning into Plasmid Vector and DNA Analysis by Restriction Enzymes

    OpenAIRE

    Elza Ibrahim Auerkari; Mamoru Ouchida; Mehmet Gunduz

    2015-01-01

    DNA cloning is one of the most important techniques In the field of molecular biology, with a critical role in analyzing the structure and function of genes and their adjacent regulatory regions. DNA cloning is helpful in learning fundamental molecular biological techniques, since DNA cloning involves a series of them, such as polymerase chain reaction (PCR), DNA ligation, bacterial transformation, bacterial culture, plasmid DNA extraction, DNA digestion with restriction enzymes and agarose g...

  19. A New Potent Inhibitor of Thrombin from the Leech Haemendipsa Yanyuanensis

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Two components of anticoagulant protein were isolated from the leech Haemendipsa yanyuanensis by heparin agarose affinity chromatography and ultracentrifugation. The determination of anticoagulant activity and characterization analysis of the pro tein using the method of chromogenic substrate indicates that the anticoagulant protein is thrombin-specific but not factor Xa-specific. The results lay a foundation for the research of the anticoagulant mechanism and application of anticoagulant protein from H. yanyua nensis.

  20. Tyrosine-specific protein kinase activity is associated with the purified insulin receptor.

    OpenAIRE

    Kasuga, M.; Fujita-Yamaguchi, Y; Blithe, D L; Kahn, C. R.

    1983-01-01

    Highly purified human placental insulin receptors were obtained by sequential affinity chromatography on wheat germ agglutinin and insulin-agarose. The preparation had an insulin binding capacity of 4,700 pmol/mg of protein approaching theoretical purity. The purified receptor revealed three major bands of Mr 135,000, 95,000, and 52,000 in NaDodSO4/polyacrylamide gel electrophoresis after reduction by dithiothreitol. All three bands were immunoprecipitated by anti-insulin-receptor antibodies....

  1. Isolation and characterization of a factor from calf serum that promotes the pigmentation of embryonic and transformed melanocytes

    OpenAIRE

    1985-01-01

    A protein (Mr = 63,000) from calf serum that promotes the pigmentation of cultured chick neural crest and mouse melanoma cells has been partially isolated and characterized in this study. The stimulation of melanin synthesis in cultured cells was used to follow its activity during purification. The pigment-promoting factor was isolated by sequential column chromatography on dye-agarose matrices followed by hydroxyapatite and high pressure molecular sieve chromatography. The factor was found t...

  2. Glycosaminoglycans from earthworms (Eisenia andrei)

    OpenAIRE

    Im, A-Rang; Park, Youmie; Sim, Joon-Soo; Zhang, Zhenqing; Liu, Zhenling; Linhardt, Robert J.; Kim, Yeong Shik

    2009-01-01

    The whole tissue of the earthworm (Eisenia andrei) was lyophilized and extracted to purify glycosaminoglycans. Fractions, eluting from an anion-exchange column at 1.0 M and 2.0 M NaCl, showed the presence of acidic polysaccharides on agarose gel electrophoresis. Monosaccharide compositional analysis showed that galactose and glucose were most abundant monosaccharides in both fractions. Depolymerization of the polysaccharide mixture with glycosaminoglycandegrading enzymes confirmed the presenc...

  3. Isolation, Affinity Purification, and Identification of Piglet Small Intestine Mucosa Receptor for Enterotoxigenic Escherichia coli K88ac+ Fimbriae

    OpenAIRE

    Fang, Lin; Gan, Zhibo; Marquardt, Ronald R.

    2000-01-01

    An affinity chromatography technique was utilized to isolate and purify the receptors of Escherichia coli K88ac+ fimbriae from the mucus of the small intestines of newborn piglets. Purified K88ac+ fimbriae were covalently immobilized onto a beaded agarose matrix (Sepharose 4B). The immobilized fimbriae were used for the affinity purification of the K88ac+ receptors. Only two major proteins were tightly and specifically bound to the immobilized fimbriae after the column containing bound recept...

  4. Purification and Characterization of Aeromonas caviae ME-1 Xylanase V, Which Produces Exclusively Xylobiose from Xylan

    OpenAIRE

    Kubata, Bruno Kilunga; Suzuki, Tohru; Horitsu, Hiroyuki; Kawai, Keiichi; Takamizawa, Kazuhiro

    1994-01-01

    A xylanase, which produces exclusively xylobiose from oat spelt and birch xylans, was isolated from the culture medium of Aeromonas caviae ME-1. The enzyme (xylanase V) was purified by ammonium sulfate fractionation, hydrophobic interaction, and ion-exchange and gel filtration chromatographies. The homogeneity of the final preparation was demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and agarose gel electrofocusing. The molecular mass and isoelectric point of the x...

  5. Contribution of chromosomal abnormalities and genes of the major histocompatibility complex to early pregnancy losses

    OpenAIRE

    Tkach I. R.; Sosnina K. O.; Huleyuk N. L.; Terpylyak O. I.; Zastavna D. V.; Weise A.; Kosyakova N.; Liehr T.

    2015-01-01

    Aim. The determination of chromosomal abnormalities in samples from early pregnancy losses and allelic polymorphism of HLA–DRB1 and DQA1 genes in couples with recurrent miscarriage. Methods. Banding cytogenetic and interphase mFISH analysis, DNA extraction by salting method, PCR, agarose gel electrophoresis. Results. Cytogenetic and molecular-cytogenetic investigations of SA material identified karyotype anomalies in 32.4 % of cases with prevalence of autosomal trisomy – 42.65 %, triploidy – ...

  6. Altered growth patterns in vitro of human papillary transitional carcinoma cells.

    OpenAIRE

    Reznikoff, C. A.; Gilchrist, K. W.; Norback, D. H.; Cummings, K. B.; ERTÜRK, E.; Bryan, G. T.

    1983-01-01

    In vitro growth patterns and morphologic characteristics of five low-grade human papillary transitional cell carcinomas (TCCs) were compared and contrasted with those of normal human urothelial cells in culture. Biopsies of TCC were performed by transurethral resection. Specimens of normal human ureters were obtained surgically. Singly dispersed TCC cells grew in 0.3% agarose semisolid medium with a cloning efficiency ranging from 0.02% to 0.71%. Singly dispersed normal ureteral urothelial ce...

  7. Effects of in vivo dexamethasone administration on in vitro bovine polymorphonuclear leukocyte function.

    OpenAIRE

    Roth, J A; Kaeberle, M L

    1981-01-01

    Polymorphonuclear leukocyte function was evaluated in vitro after in vivo administration of a single dose of dexamethasone to cattle. Purified polymorphonuclear leukocytes from dexamethasone-treated cattle displayed enhanced random migration under agarose but impaired ingestion of Staphylococcus aureus, Nitro Blue Tetrazolium reduction, chemiluminescence, iodination, and antibody-dependent, cell-mediated cytotoxicity. The depression of iodination may have been related to a drop in the proport...

  8. Transmissible mupirocin resistance in Staphylococcus aureus.

    OpenAIRE

    Rahman, M.; Noble, W. C.; Cookson, B

    1989-01-01

    The spread of two strains of Staphylococcus aureus with high level resistance to mupirocin is described. The resistance proved to be easily transferred to other S. aureus strains by filter mating experiments and on the skin of mice. No plasmid band corresponding to the resistance could be demonstrated by agarose gel electrophoresis or by caesium chloride gradient centrifugation but cleavage of 'chromosomal' DNA from resistant recipients showed bright bands of DNA absent from sensitive controls.

  9. Lateral diffusion in substrate-supported lipid monolayers as a function of ambient relative humidity.

    OpenAIRE

    Baumgart, T.; Offenhäusser, A.

    2002-01-01

    We analyzed the influence of water activity on the lateral self-diffusion of supported phospholipid monolayers. Lipid monolayer membranes were supported by polysaccharide cushions (chitosan and agarose), or glass. A simple diffusion model was derived, based on activated diffusion with an activation energy, E(a), which depends on the hydration state of the lipid headgroup. A crucial assumption of the derived model is that E(a) can be calculated assuming an exponential decay of the humidity-dep...

  10. Characterizing of Cooling Equipment for Closed Greenhouses

    OpenAIRE

    Zwart, de, H.F.; Kempkes, F.L.K.

    2008-01-01

    In order to develop new eco-sustainable technologies to set up biodegradable films for agricultural activities, spray mulching coating have been planned, prepared and tested on experimental fields. The suitable polymers used to this purpose were Arabic Gums and Agarose. Cellulose fibres were added to the polymeric water solution to improve the mulching power and to increase the tensile strength of the composite; glycerol as plasticizer was added in order to improve the mechanical response of ...

  11. Variation in sequences containing microsatellite motifs in the perennial biomass and forage grass, Phalaris arundinacea (Poaceae)

    OpenAIRE

    Barth, Susanne; Jankowska, Marta Jolanta; Hodkinson, Trevor Roland; Vellani, Tia; Klaas, Manfred

    2016-01-01

    Forty three microsatellite markers were developed for further genetic characterisation of a forage and biomass grass crop, for which genomic resources are currently scarce. The microsatellite markers were developed from a normalized EST-SSR library. All of the 43 markers gave a clear banding pattern on 3 % Metaphor agarose gels. Eight selected SSR markers were tested in detail for polymorphism across eleven DNA samples of large geographic distribution across Europe. The new set of 43 SSR mark...

  12. Self-assembled silver nanoparticles in a bow-tie antenna configuration.

    Science.gov (United States)

    Eskelinen, Antti-Pekka; Moerland, Robert J; Kostiainen, Mauri A; Törmä, Päivi

    2014-03-26

    The self-assembly of silver nanoparticles into a bow-tie antenna configuration is achieved with the DNA origami method. Instead of complicated particle geometries, spherical silver nanoparticles are used. Formation of the structures in high yields is verified with transmission electron microscopy and agarose gel electrophoresis. According to finite-difference time-domain simulations, the antenna configuration could be used as a DNA sensor. PMID:24659271

  13. Production of D-myo-inositol(1,2,4,5,6)pentakisphosphate using alginate-entrapped recombinant Pantoea agglomerans glucose-1-phosphatase

    OpenAIRE

    Ralf Greiner; Sajidan

    2008-01-01

    The glucose-1-phosphatase encoding gene (agp) of Pantoea agglomerans was sequenced and heterologously expressed in Escherichia coli. The enzyme showed very high homology to periplasmatic glucose-1-phosphatases of other members of the Enterobacteriaceae family. It was isolated from transformed Escherichia coli cells in a single step in high yields (32.3 ± 1.2 mg per litre of culture) by Ni-NT agarose affinity chromatography to >95% purity as calculated from specific activity determinations. Th...

  14. Covalently closed circular deoxyribonucleic acids in spiroplasmas.

    OpenAIRE

    Ranhand, J M; Mitchell, W. O.; Popkin, T J; Cole, R. M.

    1980-01-01

    Ten of twelve spiroplasma strains from different sources carried multiple covalently closed circular duplex deoxyribonucleic acid molecules, as shown by ethidium bromide-cesium chloride gradient centrifugation of cell lysates and examination of resulting bands by electron microscopy and agarose gel electrophoresis. Two to eight size classes per strain, comprising molecules of masses from 1 X 10(6) to 26 X 10(6), were detected. Several size classes of molecules were found in common in differen...

  15. Unambiguous typing of canine adenovirus isolates by deoxyribonucleic acid restriction-endonuclease analysis.

    OpenAIRE

    Assaf, R; Marsolais, G; Yelle, J; Hamelin, C

    1983-01-01

    Viral deoxyribonucleic acid extracted from a limited number of cells infected with canine adenovirus type 1 or type 2 was cleaved with several restriction endonucleases. Agarose gel electrophoresis of the limit digests showed stable differences between the canine adenovirus type 1 and type 2 cleavage patterns. Rapid and accurate typing of large numbers of clinical isolates may thus be done by deoxyribonucleic acid restriction-endonuclease analysis.

  16. Simple method for demonstrating small plasmid deoxyribonucleic acid molecules in oral streptococci.

    OpenAIRE

    Macrina, F L; Wood, P H; Jones, K R

    1980-01-01

    A simple procedure for rapidly demonstrating small plasmids (less than 10 megadaltons) in oral streptococci is described. Logarithmic-phase, glycine-treated cells from 1.5-ml broth cultures were converted to osmotically fragile forms and lysed with sodium dodecyl sulfate. After the hydrodynamic shearing of host chromosomal deoxyribonucleic acid, such lysates were analyzed by low-voltage agarose gel electrophoresis. Small plasmids, migrating significantly faster than chromosomal deoxyribonucle...

  17. Ex vivo model of an immobilized-enzyme reactor.

    OpenAIRE

    Bernstein, H; Langer, R

    1988-01-01

    Immobilized-enzyme reactors are beginning to be studied for a variety of therapeutic applications. To facilitate the design of these devices for different clinical situations and a diverse patient population, mathematical models may be valuable. An immobilized-heparinase (EC 4.2.2.7) reactor was selected as a model system. The device removes heparin from blood that has been anticoagulated to prevent thrombus formation. Heparinase was immobilized to cross-linked agarose particles. A mathematic...

  18. Effect of ionizing radiation on the ability of PvuII enzyme to cleave plasmid DNA

    International Nuclear Information System (INIS)

    Restriction endonucleases type II are enzymes which recognize palindromic DNA base sequences and specifically cleave the double-stranded DNA at the recognition sites. The ability of PvuII enzyme irradiated by gamma radiation to cleave DNA plasmid pcDNA3 was examined by the agarose electrophoresis method. Doses above 200 Gy reduced the enzyme's recognition efficiency for specific DNA base sequences and doses above 500 Gy inactivated the enzyme completely. (orig.)

  19. Hevea brasiliensis cell suspension peroxidase: purification, characterization and application for dye decolorization

    OpenAIRE

    Chanwun, Thitikorn; Muhamad, Nisaporn; Chirapongsatonkul, Nion; Churngchow, Nunta

    2013-01-01

    Peroxidases are oxidoreductase enzymes produced by most organisms. In this study, a peroxidase was purified from Hevea brasiliensis cell suspension by using anion exchange chromatography (DEAE-Sepharose), affinity chromatography (Con A-agarose) and preparative SDS-PAGE. The obtained enzyme appeared as a single band on SDS-PAGE with molecular mass of 70 kDa. Surprisingly, this purified peroxidase also had polyphenol oxidase activity. However, the biochemical characteristics were only studied i...

  20. Long range restriction analysis of the bovine casein genes.

    OpenAIRE

    Ferretti, L; Leone, P.; Sgaramella, V

    1990-01-01

    Pulsed field gel electrophoresis (PFGE) was used to analyse the organization of the bovine alpha s1, alpha s2, beta and kappa casein genes. High molecular weight DNA was prepared from fibroblasts and lymphocytes embedded in agarose and was digested with the restriction endonucleases Clal, Sall, Smal, Xhol. The digestion products were separated by PFGE, transfered to nitrocellulose filters and hybridized to probes corresponding to the cDNAs of the four bovine casein genes. The casein genes wer...

  1. REMOVAL OF SYNTHETIC DYE BASIC VIOLET 3 BY IMMOBILISED CANDIDA TROPICALIS GROWN ON SUGARCANE BAGASSE EXTRACT MEDIUM

    OpenAIRE

    CHARUMATHI D; NILANJANA DAS

    2010-01-01

    The removal of synthetic dye Basic Violet 3 using immobilised yeast Candida tropicalis grown on sugarcane bagasse extract medium was investigated. The various immobilization matrices viz. carboxymethyl cellulose, sodium alginate, agar, agarose and polyvinyl alcohol were tested and highest dye removal efficiency (99%) was noted in sodium alginate immobilised beads. The concentration of sodium alginate, bead size and cell concentration were optimized as 3%, 2mm and 3x 106 cells/g bead respectiv...

  2. Scaffold- and Cell System-Based Bone Grafts in Tissue Engineering (Review)

    OpenAIRE

    Kuznetsova D.S.; Timashev P.S.; Bagratashvili V.N.; Zagaynova Е.V.

    2014-01-01

    The review considers the current trends in tissue engineering including maxillofacial surgery based on the use of scaffolds, autologous stem cells and bioactive substances. The authors have shown the advantages and disadvantages of basic materials used for scaffold synthesis — three-dimensional porous or fiber matrices serving as a mechanical frame for cells; among such materials there are natural polymers (collagen, cellulose, fibronectin, chitosan, alginate and agarose, fibroin), synthetic ...

  3. Isolation and characterization of a novel ribonucleoprotein particle: large structures contain a single species of small RNA

    OpenAIRE

    1986-01-01

    Rat liver coated vesicle preparations were frequently found to contain small ovoid bodies, which resembled coated vesicles in morphology. We have purified these bodies to homogeneity using sucrose density gradients and preparative agarose gel electrophoresis. When negatively stained and viewed by electron microscopy, the purified structures display a very distinct and complex morphology, resembling the multiple arches which form cathedral vaults. They measure 35 X 65 nm and are therefore cons...

  4. DNA DAMAGE QUANTITATION BY ALKALINE GEL ELECTROPHORESIS.

    Energy Technology Data Exchange (ETDEWEB)

    SUTHERLAND,B.M.; BENNETT,P.V.; SUTHERLAND, J.C.

    2004-03-24

    Physical and chemical agents in the environment, those used in clinical applications, or encountered during recreational exposures to sunlight, induce damages in DNA. Understanding the biological impact of these agents requires quantitation of the levels of such damages in laboratory test systems as well as in field or clinical samples. Alkaline gel electrophoresis provides a sensitive (down to {approx} a few lesions/5Mb), rapid method of direct quantitation of a wide variety of DNA damages in nanogram quantities of non-radioactive DNAs from laboratory, field, or clinical specimens, including higher plants and animals. This method stems from velocity sedimentation studies of DNA populations, and from the simple methods of agarose gel electrophoresis. Our laboratories have developed quantitative agarose gel methods, analytical descriptions of DNA migration during electrophoresis on agarose gels (1-6), and electronic imaging for accurate determinations of DNA mass (7-9). Although all these components improve sensitivity and throughput of large numbers of samples (7,8,10), a simple version using only standard molecular biology equipment allows routine analysis of DNA damages at moderate frequencies. We present here a description of the methods, as well as a brief description of the underlying principles, required for a simplified approach to quantitation of DNA damages by alkaline gel electrophoresis.

  5. Electroeluting DNA fragments.

    Science.gov (United States)

    Zarzosa-Alvarez, Ana L; Sandoval-Cabrera, Antonio; Torres-Huerta, Ana L; Bermudez-Cruz, Rosa M

    2010-01-01

    Purified DNA fragments are used for different purposes in Molecular Biology and they can be prepared by several procedures. Most of them require a previous electrophoresis of the DNA fragments in order to separate the band of interest. Then, this band is excised out from an agarose or acrylamide gel and purified by using either: binding and elution from glass or silica particles, DEAE-cellulose membranes, "crush and soak method", electroelution or very often expensive commercial purification kits. Thus, selecting a method will depend mostly of what is available in the laboratory. The electroelution procedure allows one to purify very clean DNA to be used in a large number of applications (sequencing, radiolabeling, enzymatic restriction, enzymatic modification, cloning etc). This procedure consists in placing DNA band-containing agarose or acrylamide slices into sample wells of the electroeluter, then applying current will make the DNA fragment to leave the agarose and thus be trapped in a cushion salt to be recovered later by ethanol precipitation. PMID:20834225

  6. Preparation of high-molecular-weight DNA from Drosophila embryos.

    Science.gov (United States)

    Karpen, Gary H

    2009-07-01

    Standard methods for extracting DNA from cells or organisms (e.g., phenol extraction and ethanol precipitation) produce fragments with an average size of 50-200 kb under optimal conditions. The shearing forces that are applied to DNA in solution during mechanical vortexing or mixing and pipetting produce frequent double-stranded breaks. To prepare high-molecular-weight (HMW) DNA, it is necessary to guard against such damaging forces by performing all extractions and manipulations on DNA that is embedded within a protective matrix. Preparation of HMW DNA from Drosophila embryos is described in detail here because, in our hands, it is the simplest and most reliable protocol and can be used for large- or small-scale preparations. The overall strategy is to purify nuclei, gently embed them in molten agarose, and then extract proteins and perform other enzymatic reactions by transferring the solidified agarose block into the appropriate solutions. Salts, soaps, and enzymes act on the DNA by diffusing through the agarose matrix, while the matrix protects the DNA from shearing forces. PMID:20147219

  7. Electrophoretic mobility as a tool to separate immune adjuvant saponins from Quillaja saponaria Molina.

    Science.gov (United States)

    Gilabert-Oriol, Roger; Weng, Alexander; von Mallinckrodt, Benedicta; Stöshel, Anja; Nissi, Linda; Melzig, Matthias F; Fuchs, Hendrik; Thakur, Mayank

    2015-06-20

    Quillaja saponins are used as adjuvants in animal vaccines but their application in human vaccination is still under investigation. Isolation and characterization of adjuvant saponins is very tedious. Furthermore, standardization of Quillaja saponins is critical pertaining to its application in humans. In this study, a convenient method based on agarose gel electrophoresis was developed for the separation of Quillaja saponins. Six different commercial Quillaja saponins were segregated by size/charge into numerous fractions. Each of the fractions was characterized by ESI-TOF-MS spectroscopy and thin layer chromatography. Real-time impedance-based monitoring and red blood cell lysis assay were used to evaluate cytotoxicity and hemolytic activities respectively. Two specific regions in the agarose gel (delimited by specific relative electrophoretic mobility values) were identified and characterized by exclusive migration of acylated saponins known to possess immune adjuvant properties (0.18-0.58), and cytotoxic and hemolytic saponins (0.18-0.94). In vivo experiments in mice with the isolated fractions for evaluation of adjuvant activity also correlated with the relative electrophoretic mobility. In addition to the separation of specific Quillaja saponins with adjuvant effects as a pre-purification step to HPLC, agarose gel electrophoresis stands out as a new method for rapid screening, separation and quality control of saponins. PMID:25839418

  8. Fabricating neuromast-inspired gel structures for membrane-based hair cell sensing

    Science.gov (United States)

    Tamaddoni, Nima J.; Stephens, Christopher P.; Sarles, S. A.

    2012-04-01

    Recent research has shown that a new class of mechanical sensor, assembled from biomolecules and which features an artificial cell membrane as the sensing element, can be used to mimic basic hair cell mechanotransduction in vertebrates. The work presented in this paper is motivated by the need to increase sensor performance and stability by refining the methods used to fabricate and connect lipid-encapsulated hydrogels. Inspired by superficial neuromasts found on fish, three hydrogel materials are compared for their ability to be readily shaped into neuromast-inspired geometries and enable lipid bilayer formation using self-assembly at an oil/water interface. Agarose, polyethylene glycol (PEG, 6kg/mole), and hydroxyethyl methacrylate (HEMA) gel materials are compared. The results of this initial study determined that UV-curable gel materials such as PEG and HEMA enable more accurate shaping of the gel-needed for developing a sensor that uses a gel material both for mechanical support and membrane formation-compared to agarose. However, the lower hydrophobicity of agarose and PEG materials provide a more fluid, water-like environment for membrane formation-unlike HEMA. In working toward a neuromast-inspired design, a final experiment demonstrates that a bilayer can also be formed directly between two lipid-covered PEG surfaces. These initial results suggest that candidate gel materials with a low hydrophobicity, high fluidity, and a low modulus can be used to provide membrane support.

  9. High ionic strength narrows the population of sites participating in protein ion-exchange adsorption: a single-molecule study.

    Science.gov (United States)

    Kisley, Lydia; Chen, Jixin; Mansur, Andrea P; Dominguez-Medina, Sergio; Kulla, Eliona; Kang, Marci K; Shuang, Bo; Kourentzi, Katerina; Poongavanam, Mohan-Vivekanandan; Dhamane, Sagar; Willson, Richard C; Landes, Christy F

    2014-05-23

    The retention and elution of proteins in ion-exchange chromatography is routinely controlled by adjusting the mobile phase salt concentration. It has repeatedly been observed, as judged from adsorption isotherms, that the apparent heterogeneity of adsorption is lower at more-eluting, higher ionic strength. Here, we present an investigation into the mechanism of this phenomenon using a single-molecule, super-resolution imaging technique called motion-blur Points Accumulation for Imaging in Nanoscale Topography (mbPAINT). We observed that the number of functional adsorption sites was smaller at high ionic strength and that these sites had reduced desorption kinetic heterogeneity, and thus narrower predicted elution profiles, for the anion-exchange adsorption of α-lactalbumin on an agarose-supported, clustered-charge ligand stationary phase. Explanations for the narrowing of the functional population such as inter-protein interactions and protein or support structural changes were investigated through kinetic analysis, circular dichroism spectroscopy, and microscopy of agarose microbeads, respectively. The results suggest the reduction of heterogeneity is due to both electrostatic screening between the protein and ligand and tuning the steric availability within the agarose support. Overall, we have shown that single molecule spectroscopy can aid in understanding the influence of ionic strength on the population of functional adsorbent sites participating in the ion-exchange chromatographic separation of proteins. PMID:24751557

  10. Visualization of DNA molecules in time during electrophoresis

    Science.gov (United States)

    Lubega, Seth

    1991-01-01

    For several years individual DNA molecules have been observed and photographed during agarose gel electrophoresis. The DNA molecule is clearly the largest molecule known. Nevertheless, the largest molecule is still too small to be seen using a microscope. A technique developed by Morikawa and Yanagida has made it possible to visualize individual DNA molecules. When these long molecules are labeled with appropriate fluorescence dyes and observed under a fluorescence microscope, although it is not possible to directly visualize the local ultrastructure of the molecules, yet because they are long light emitting chains, their microscopic dynamical behavior can be observed. This visualization works in the same principle that enables one to observe a star through a telescope because it emits light against a dark background. The dynamics of individual DNA molecules migrating through agarose matrix during electrophoresis have been described by Smith et al. (1989), Schwartz and Koval (1989), and Bustamante et al. (1990). DNA molecules during agarose gel electrophoresis advance lengthwise thorough the gel in an extended configuration. They display an extension-contraction motion and tend to bunch up in their leading ends as the 'heads' find new pores through the gel. From time to time they get hooked on obstacles in the gel to form U-shaped configurations before they resume their linear configuration.

  11. Transient magnetic birefringence for determining magnetic nanoparticle diameters in dense, highly light scattering media

    Science.gov (United States)

    Köber, Mariana; Moros, Maria; Grazú, Valeria; de la Fuente, Jesus M.; Luna, Mónica; Briones, Fernando

    2012-04-01

    The increasing use of biofunctionalized magnetic nanoparticles in biomedical applications calls for further development of characterization tools that allow for determining the interactions of the nanoparticles with the biological medium in situ. In cell-incubating conditions, for example, nanoparticles may aggregate and serum proteins adsorb on the particles, altering the nanoparticles’ performance and their interaction with cell membranes. In this work we show that the aggregation of spherical magnetite nanoparticles can be detected with high sensitivity in dense, highly light scattering media by making use of magnetically induced birefringence. Moreover, the hydrodynamic particle diameter distribution of anisometric nanoparticle aggregates can be determined directly in these media by monitoring the relaxation time of the magnetically induced birefringence. As a proof of concept, we performed measurements on nanoparticles included in an agarose gel, which scatters light in a similar way as a more complex biological medium but where particle-matrix interactions are weak. Magnetite nanoparticles were separated by agarose gel electrophoresis and the hydrodynamic diameter distribution was determined in situ. For the different particle functionalizations and agarose concentrations tested, we could show that gel electrophoresis did not yield a complete separation of monomers and small aggregates, and that the electrophoretic mobility of the aggregates decreased linearly with the hydrodynamic diameter. Furthermore, the rotational particle diffusion was not clearly affected by nanoparticle-gel interactions. The possibility to detect nanoparticle aggregates and their hydrodynamic diameters in complex scattering media like cell tissue makes transient magnetic birefringence an interesting technique for biological applications.

  12. Effects of estrogen on very low-density lipoprotein triglyceride metabolism in fed and fasted chicks

    International Nuclear Information System (INIS)

    A single injection of estrogen into growing chicks resulted in a marked elevation in plasma triglyceride (TG) followed by phospholipid (PL) and cholesterol (CH) in both fed and fasted chicks. Estrogen caused a development of massive fatty liver in fed chicks. Hepatic malic enzyme and glucose-6-phosphate dehydrogenase activities also increased significantly in fed chicks and, to a small extent, in fasted chicks. Very low density lipoproteins (VLDL) were barely detectable in the fasted control plasma. However, the VLDL concentration increased markedly upon estrogen injection, becoming the most prevalent lipoprotein in the plasma. The administration of estrogen resulted in an increase in oleic acid and a decrease in linoleic acid content except in the cholesteryl ester of VLDL and LDL. VLDL of estrogenized birds had β-mobility on agarose gel electrophoresis, and they eluted in two peaks on agarose gel filtration chromatography. Both peaks on gel filtration exhibited the same β-mobility on agarose gel electrophoresis. Nevertheless, the apoprotein composition of these two peaks were substantially different from each other; apo B was not present in the first peak VLDL. VLDL-TG kinetic studies conducted in vivo, using 14C-TG-VLDL prepared endogenously from control and estrogenized chicks revealed that VLDL-TG produced from the former had a higher fractional catabolic rate (FCR) than VLDL-TG from the latter

  13. Lymphocyte receptors for pertussis toxin

    International Nuclear Information System (INIS)

    We have investigated human T-lymphocyte receptors for pertussis toxin by affinity isolation and photoaffinity labeling procedures. T lymphocytes were obtained from peripheral human blood, surface iodinated, and solubilized in Triton X-100. The iodinated mixture was then passed through pertussis toxin-agarose, and the fractions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Autoradiography of the fixed, dried gels revealed several bands in the pertussis toxin-bound fraction that were not observed in fractions obtained from histone or fetuin-agarose. Further investigations employed a photoaffinity labeling reagent, sulfosuccinimidyl 2-(p-azido-salicylamido)-1,3'-dithiopropionate, to identify pertussis toxin receptors in freshly isolated peripheral blood monocytic cells, T lymphocytes, and Jurkat cells. In all three cell systems, the pertussis toxin affinity probe specifically labeled a single protein species with an apparent molecular weight of 70,000 that was not observed when the procedure was performed in the presence of excess unmodified pertussis toxin. A protein comparable in molecular weight to the one detected by the photoaffinity labeling technique was also observed among the species that bound to pertussis toxin-agarose. The results suggest that pertussis toxin may bind to a 70,000-Da receptor in human T lymphocytes

  14. Transient magnetic birefringence for determining magnetic nanoparticle diameters in dense, highly light scattering media

    International Nuclear Information System (INIS)

    The increasing use of biofunctionalized magnetic nanoparticles in biomedical applications calls for further development of characterization tools that allow for determining the interactions of the nanoparticles with the biological medium in situ. In cell-incubating conditions, for example, nanoparticles may aggregate and serum proteins adsorb on the particles, altering the nanoparticles’ performance and their interaction with cell membranes. In this work we show that the aggregation of spherical magnetite nanoparticles can be detected with high sensitivity in dense, highly light scattering media by making use of magnetically induced birefringence. Moreover, the hydrodynamic particle diameter distribution of anisometric nanoparticle aggregates can be determined directly in these media by monitoring the relaxation time of the magnetically induced birefringence. As a proof of concept, we performed measurements on nanoparticles included in an agarose gel, which scatters light in a similar way as a more complex biological medium but where particle–matrix interactions are weak. Magnetite nanoparticles were separated by agarose gel electrophoresis and the hydrodynamic diameter distribution was determined in situ. For the different particle functionalizations and agarose concentrations tested, we could show that gel electrophoresis did not yield a complete separation of monomers and small aggregates, and that the electrophoretic mobility of the aggregates decreased linearly with the hydrodynamic diameter. Furthermore, the rotational particle diffusion was not clearly affected by nanoparticle–gel interactions. The possibility to detect nanoparticle aggregates and their hydrodynamic diameters in complex scattering media like cell tissue makes transient magnetic birefringence an interesting technique for biological applications. (paper)

  15. DNA unwinding induced by photoaddition of psoralen derivatives and determination of dark-binding equilibrium constants by gel electrophoresis

    International Nuclear Information System (INIS)

    Derivatives of furo[3,2-g]coumarin (psoralen) can bind to the DNA double helix and, in the presence of long-wavelength uv light, the bound psoralen may react covalently with pyrimidine residues on one or both strands of the helix. By using agarose gel electrophoresis, we have determined the unwinding angle associated with each of four different psoralen derivatives to be 280 +- 40. For 4,5',8-trimethylpsoralen (trioxsalen) the unwinding angle was found to be independent of the initial DNA superhelix density in the range that is accessible to agarose gel electrophoresis. Also by using agarose gel electrophoresis, we have determined the unwinding angle for ethidium intercalation. This was done by the total relaxation of supercoiled DNA in the presence of a series of ethidium concentrations. By using published values for the association constant for ethidium binding to DNA and evaluating the final superhelix density (after removal of ethidium) of the DNA on gels, we calculated an unwinding angle of 290 +- 30. Assuming an unwinding angle of 280 for the noncovalent intercalation of psoralen derivatives, we used the same procedure to determine intercalation binding constants. The association constants for 4'-aminomethyltrioxsalen were 300 to 1400 M-1 in NaCl at 0.2 to 0.05 M and 300 to 2500 M-1 in Mg2+ at 4 to 0.5 mM. The association constant for 4'-hydroxymethyltrioxsalen in 0.5 mM Mg2+ was determined to be 70 M-1

  16. Isolation and Identification of Virus dsRNA from Strawberry Plants

    Institute of Scientific and Technical Information of China (English)

    LI He; DAI Hong-yan; ZHANG Zhi-hong; GAO Xiu-yan; DU Guo-dong; ZHANG Xin-yu

    2007-01-01

    The analysis of virus genome is based on nucleic acid isolation. The aims of this study were to develop a method for isolation and identification of virus double-stranded ribonucleic acid (dsRNA) and to elucidate the nucleotide sequences of strawberry virus. Using the modified method, virus dsRNA was extracted from strawberry virus indicator plants and cultivated strawberry plants and detected using agarose gel electrophoresis with ethidium bromide staining and reverse transcription-polymerase chain reaction (RT-PCR). The quantity of virus dsRNA varied among strawberry cultivars. The quantity of dsRNA from in vitro plantlets was higher than that from the young leaves of field plants. For the field-grown plants, there was more dsRNA in the young leaves. Virus dsRNA extracted from strawberry plants was resistant to deoxyribonuclease Ⅰ (DNase Ⅰ ), but evidently, it became resistant to ribonuclease A (RNase A) only in the presence of 0.5 M NaCl. Its bands in agarose gel could be readily recycled using an agarose gel DNA purification kit. With RT-PCR, the segments of both strawberry mottle virus and Strawberry mild yellow edge virus genomes were amplified by using the virus dsRNA recycled from gel or treated with DNase Ⅰ /RNase A as templates. The system developed for dsRNA isolation and identification in strawberry plants laid a sound foundation for the work on genome analysis of strawberry virus isolates in China.

  17. Fabrication of microlens array on silicon surface using electrochemical wet stamping technique

    Science.gov (United States)

    Lai, Lei-Jie; Zhou, Hang; Zhu, Li-Min

    2016-02-01

    This paper focuses on the fabrication of microlens array (MLA) on silicon surface by taking advantage of a novel micromachining approach, the electrochemical we stamping (E-WETS). The E-WETS allows the direct imprinting of MLA on an agarose stamp into the substrate through a selective anodic dissolution process. The pre-patterned agarose stamp can direct and supply the solution preferentially on the contact area between the agarose stamp and the substrate, to which the electrochemical reaction is confined. The anodic potential vs. saturated calomel electrode is optimized and 1.5 V is chosen as the optimum value for the electrochemical polishing of p-Si. A refractive MLA on a PMMA mold is successfully transferred onto the p-Si surface. The machining deviations of the fabricated MLA from those on the mold are 0.44% in diameter and 2.1% in height respectively, and the machining rate in HF is around 1.1 μm/h. The surface roughness of the fabricated MLA is less than 12 nm owing to the electrochemical polishing process. The results demonstrate that E-WETS is a promising approach to fabricate MLA on p-Si surface with high accuracy and efficiency.

  18. Peptides and proteins in a confined environment: NMR spectra at natural isotopic abundance.

    Science.gov (United States)

    Pastore, Annalisa; Salvadori, Severo; Temussi, Piero Andrea

    2007-05-01

    Confinement of proteins and peptides in a small inert space mimics the natural environment of the cell, allowing structural studies in conditions that stabilize folded conformations. We have previously shown that confinement in polyacrylamide gels (PAGs) is sufficient to induce a change in the viscosity of the aqueous solution without changing the composition and temperature of the solvent. The main limitation of a PAG to run NMR experiments in a confined environment is the need for labelling the peptides. Here we report the use of the agarose gel to run the NMR spectra of proteins and peptides. We show that agarose gels are completely transparent in NMR experiments, relieving the need for labelling. Although it is necessary to expose biomolecules to fairly high temperatures during sample preparation, we believe that this is not generally an obstacle to the study of peptides, and found that the method is also compatible with temperature-resistant proteins. The mesh of agarose gels is too wide for direct effects of confinement on the stability of proteins but confinement can be easily exploited to interact the proteins with other reagents, including crowding macromolecules that can eventually lead to fold stabilization. The use of these gels is ideally suited for low-temperature studies; we show that a very flexible peptide at subzero temperatures is stabilized into a well-folded conformation. PMID:17436341

  19. Solid phase group specific absorbants in assays for glycoproteins

    International Nuclear Information System (INIS)

    The focus of this paper is on several technical advances in the assays for glycoprotein hormones and enzymes that have been achieved by use of the solid phase carbohydrate specific adsorbant concanavalin-A. Puriffication of glycoprotein radioligand after labelling by the chloramine-T method is readily accomplished using a small column of agarose bound concanavalin-A which separates glycoprotein radioligand from radioiodide and radiolabelled unadsorbed contaminants. After concanavalin-A column chromatography, radiolabelled glycoprotein hormone preparations exhibited improved binding to antibodies and tissue receptors. To increase the effective sensitivity of radioimmunoassays for glycoproteins, agarose bound concanavalin-A is used to extract and concentrate the glycoproteins from various biologic samples. For example, the effective sensitivity for the detection of human thyrotropin in serum was improved approximately 5 fold by using concanavalin-A concentrates of 1.5 ml of serum. Partial purification of the glycoprotein dopamine-β-hydroxylase from serum using agarose bound concanavalin-A resulted in separation of the serum factors that interfere with the measurement of enzyme activity. We conclude that in assays for glycoproteins, concanavalin-A is useful for purification of radioligand, for preparation of concentrates of glycoproteins from biologic samples, and for separation of glycoproteins from various interfering factors contained in biologic samples prior to radioligand or radioenzyme assay. (orig.)

  20. Solid-phase group-specific adsorbants in assays for glycoproteins

    International Nuclear Information System (INIS)

    The focus of the paper is on several technical advances in the assays for glycoprotein hormones and enzymes that have been achieved by the use of the solid-phase cabohydrate-specific adsorbant concanavalin-A. Purification of glycoprotein radioligand after labelling by the Chloramine-T method is readily accomplished using a small column of agarose-bound concanavalin-A which separates glycoprotein radioligand from radioiodide and radiolabelled unadsorbed contaminants. After concanavalin-A column chromatography, radiolabelled glycoprotein hormone preparations exhibited improved binding to antibodies and tissue receptors. To increase the effective sensitivity of radioimmunoassays for glycoproteins, agarose-bound concanavalin-A is used to extract and concentrate the glycoproteins from various biological samples. For example, the effective sensitivity for the detection of human thyrotropin in serum was improved approximately 5-fold by using concanavalin-A concentrates of 1.5ml of serum. Partial purification of the glycoprotein dopamine-β-hydroxylase from serum using agarose-bound concanavalin-A resulted in separation of the serum factors that interfere with the measurement of enzyme activity. We conclude that in assays for glycoproteins, concanavalin-A is useful for purification of radioligand, for preparation of concentrates of glycoproteins from biological samples and for separation of glycoproteins from various interfering factors contained in biological samples before radioligand or radioenzyme assay. (author)

  1. Stabilization by multipoint covalent attachment of a biocatalyst with polygalacturonase activity used for juice clarification.

    Science.gov (United States)

    Ramírez Tapias, Yuly A; Rivero, Cintia W; Gallego, Fernando López; Guisán, José M; Trelles, Jorge A

    2016-10-01

    Derivatized-agarose supports are suitable for enzyme immobilization by different methods, taking advantage of different physical, chemical and biological conditions of the protein and the support. In this study, agarose particles were modified with MANAE, PEI and glyoxyl groups and evaluated to stabilize polygalacturonase from Streptomyces halstedii ATCC 10897. A new immobilized biocatalyst was developed using glyoxyl-agarose as support; it exhibited high performance in degrading polygalacturonic acid and releasing oligogalacturonides. Maximal enzyme activity was detected at 5h of reaction using 0.05g/mL of immobilized biocatalyst, which released 3mg/mL of reducing sugars and allowed the highest product yield conversion and increased stability. These results are very favorable for pectin degradation with reusability up to 18 successive reactions (90h) and application in juice clarification. Plum (4.7°Bx) and grape (10.6°Bx) juices were successfully clarified, increasing reducing sugars content and markedly decreasing turbidity and viscosity. PMID:27132847

  2. The use of caspase inhibitors in pulsed-field gel electrophoresis may improve the estimation of radiation-induced DNA repair and apoptosis

    International Nuclear Information System (INIS)

    Radiation-induced DNA double-strand break (DSB) repair can be tested by using pulsed-field gel electrophoresis (PFGE) in agarose-encapsulated cells. However, previous studies have reported that this assay is impaired by the spontaneous DNA breakage in this medium. We investigated the mechanisms of this fragmentation with the principal aim of eliminating it in order to improve the estimation of radiation-induced DNA repair. Samples from cancer cell cultures or xenografted tumours were encapsulated in agarose plugs. The cell plugs were then irradiated, incubated to allow them to repair, and evaluated by PFGE, caspase-3, and histone H2AX activation (γH2AX). In addition, apoptosis inhibition was evaluated through chemical caspase inhibitors. We confirmed that spontaneous DNA fragmentation was associated with the process of encapsulation, regardless of whether cells were irradiated or not. This DNA fragmentation was also correlated to apoptosis activation in a fraction of the cells encapsulated in agarose, while non-apoptotic cell fraction could rejoin DNA fragments as was measured by γH2AX decrease and PFGE data. We were able to eliminate interference of apoptosis by applying specific caspase inhibitors, and improve the estimation of DNA repair, and apoptosis itself. The estimation of radiation-induced DNA repair by PFGE may be improved by the use of apoptosis inhibitors. The ability to simultaneously determine DNA repair and apoptosis, which are involved in cell fate, provides new insights for using the PFGE methodology as functional assay

  3. In-house preparation of hydrogels for batch affinity purification of glutathione S-transferase tagged recombinant proteins

    Directory of Open Access Journals (Sweden)

    Buhrman Jason S

    2012-09-01

    Full Text Available Abstract Background Many branches of biomedical research find use for pure recombinant proteins for direct application or to study other molecules and pathways. Glutathione affinity purification is commonly used to isolate and purify glutathione S-transferase (GST-tagged fusion proteins from total cellular proteins in lysates. Although GST affinity materials are commercially available as glutathione immobilized on beaded agarose resins, few simple options for in-house production of those systems exist. Herein, we describe a novel method for the purification of GST-tagged recombinant proteins. Results Glutathione was conjugated to low molecular weight poly(ethylene glycol diacrylate (PEGDA via thiol-ene “click” chemistry. With our in-house prepared PEGDA:glutathione (PEGDA:GSH homogenates, we were able to purify a glutathione S-transferase (GST green fluorescent protein (GFP fusion protein (GST-GFP from the soluble fraction of E. coli lysate. Further, microspheres were formed from the PEGDA:GSH hydrogels and improved protein binding to a level comparable to purchased GSH-agarose beads. Conclusions GSH containing polymers might find use as in-house methods of protein purification. They exhibited similar ability to purify GST tagged proteins as purchased GSH agarose beads.

  4. Quantification of specific bindings of biomolecules by magnetorelaxometry

    Directory of Open Access Journals (Sweden)

    Steinhoff Uwe

    2008-03-01

    Full Text Available Abstract The binding reaction of the biomolecules streptavidin and anti-biotin antibody, both labelled by magnetic nanoparticles (MNP, to biotin coated on agarose beads, was quantified by magnetorelaxometry (MRX. Highly sensitive SQUID-based MRX revealed the immobilization of the MNP caused by the biotin-streptavidin coupling. We found that about 85% of streptavidin-functionalised MNP bound specifically to biotin-agarose beads. On the other hand only 20% of antibiotin-antibody functionalised MNP were specifically bound. Variation of the suspension medium revealed in comparison to phosphate buffer with 0.1% bovine serum albumin a slight change of the binding behaviour in human serum, probably due to the presence of functioning (non heated serum proteins. Furthermore, in human serum an additional non-specific binding occurs, being independent from the serum protein functionality. The presented homogeneous bead based assay is applicable in simple, uncoated vials and it enables the assessment of the binding kinetics in a volume without liquid flow. The estimated association rate constant for the MNP-labelled streptavidin is by about two orders of magnitude smaller than the value reported for free streptavidin. This is probably due to the relatively large size of the magnetic markers which reduces the diffusion of streptavidin. Furthermore, long time non-exponential kinetics were observed and interpreted as agglutination of the agarose beads.

  5. Visualization of yeast chromosomal DNA

    Science.gov (United States)

    Lubega, Seth

    1990-01-01

    The DNA molecule is the most significant life molecule since it codes the blue print for other structural and functional molecules of all living organisms. Agarose gel electrophoresis is now being widely used to separate DNA of virus, bacteria, and lower eukaryotes. The task was undertaken of reviewing the existing methods of DNA fractionation and microscopic visualization of individual chromosonal DNA molecules by gel electrophoresis as a basis for a proposed study to investigate the feasibility of separating DNA molecules in free fluids as an alternative to gel electrophoresis. Various techniques were studied. On the molecular level, agarose gel electrophoresis is being widely used to separate chromosomal DNA according to molecular weight. Carl and Olson separate and characterized the entire karyotype of a lab strain of Saccharomyces cerevisiae. Smith et al. and Schwartz and Koval independently reported the visualization of individual DNA molecules migrating through agarose gel matrix during electrophoresis. The techniques used by these researchers are being reviewed in the lab as a basis for the proposed studies.

  6. Purification and characterization of ethylene inducing proteins from cellulysin.

    Science.gov (United States)

    Fuchs, Y; Anderson, J D

    1987-07-01

    Ethylene inducing proteins were partially purified and characterized from the cell wall digesting enzyme mixture, Cellulysin. Purification included binding to Sephacryl S-200, isoelectric focusing, molecular sieving on Sephadex G-75, agarose electrophoresis, and sizing using a Superose 12 column. At least three active proteins were obtained from the Sephadex G-75 fraction that move towards the cathode during nondenaturing agarose electrophoresis. These three protein fractions separated by preparative agarose electrophoresis contain polypeptide patterns that are very similar on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The fractions contain three main Coomassie blue stained bands of about 10, 14, and 18 kilodaltons. Gel filtration of the major fraction on a Superose 12 column yields an active peak with an apparent molecular weight of 27,000. Proteolytic enzymes, in the presence of urea, destroy the ethylene inducing activity. We conclude that the ethylene inducing factor (EIF) that we have isolated from Cellulysin is protein. Similar ethylene inducing factors are present in Cellulase RS. Ethylene inducing components from pectinase, Pectolyase, and Rhozyme do not bind to Sephacryl like EIF from Cellulysin. Thus, the components responsible for the ethylene inducing activity in these latter enzyme preparations differ from that of EIF. PMID:16665512

  7. DISPOSITIVOS DGT MODIFICADOS COM MATERIAIS ALTERNATIVOS PARA USO NA ESPECIAÇÃO DE ELEMENTOS TRAÇO

    Directory of Open Access Journals (Sweden)

    Cristiano L. Chostak

    2015-03-01

    Full Text Available The use of MT-K10 Montmorillonite immobilized onto agarose was investigated in this work as an alternative binding phase in Diffusive Gradient in Thin Film (DGT devices for the determination of metallic labile species. In addition, agarose itself was also used as the diffusive phase. The percentage of sorption of Zn2+, Cu2+, Cr3+, Mn2+, Cd2+, Pb2+, and Ni2+ onto the binding phase was higher than 80% and the desorption process for all elements was also greater than 75%. Elution factors were determined experimentally, ranging from 0.74 for Zn2+ and 0.90 for Cr3+ and Pb2+. The accumulation of all species was linear with time, in agreement with the Fick's 1st law of diffusion. The deployment of the alternative devices in natural waters was compared to commercial devices. Labile concentrations determined by the alternative devices were slightly superior compared to results obtained with the deployment of original DGT devices due to the less restrictive pores of agarose.

  8. How to assess the plasma delivery of RONS into tissue fluid and tissue

    Science.gov (United States)

    Oh, Jun-Seok; Szili, Endre J.; Gaur, Nishtha; Hong, Sung-Ha; Furuta, Hiroshi; Kurita, Hirofumi; Mizuno, Akira; Hatta, Akimitsu; Short, Robert D.

    2016-08-01

    The efficacy of helium (He) and argon (Ar) plasma jets are being investigated for different healthcare applications including wound and cancer therapy, sterilisation and surface disinfections. Current research points to a potential link between the generation of reactive oxygen and nitrogen species (RONS) and outcomes in a range of biological and medical applications. As new data accrue, further strengthening this link, it becomes important to understand the controlled delivery of RONS into solutions, tissue fluids and tissues. This paper investigates the use of He and Ar plasma jets to deliver three RONS (hydrogen peroxide—H2O2, nitrite—\\text{NO}2- and nitrate—\\text{NO}3- ) and molecular oxygen (O2) directly into deionised (DI) water, or indirectly into DI water through an agarose target. The DI water is used in place of tissue fluid and the agarose target serves as a surrogate of tissue. Direct plasma jet treatments deliver more RONS and O2 than the through-agarose treatments for equivalent treatments times. The former only deliver RONS whilst the plasma jets are ignited; the latter continues to deliver RONS into the DI water long after the plasmas are extinguished. The He plasma jet is more effective at delivering H2O2 and \\text{NO}2- directly into DI water, but the Ar plasma jet is more effective at nitrating the DI water in both direct and through-agarose treatments. DI water directly treated with the plasma jets is deoxygenated, with the He plasma jet purging more O2 than the Ar plasma jet. This effect is known as ‘sparging’. In contrast, for through-agarose treatments both jets oxygenated the DI water. These results indicate that in the context of direct and indirect plasma jet treatments of real tissue fluids and tissue, the choice of process gas (He or Ar) could have a profound effect on the concentrations of RONS and O2. Irrespective of operating gas, sparging of tissue fluid (in an open wound) for long prolonged periods during direct plasma

  9. Near net shape forming processes for chemically prepared zinc oxide varistors.

    Energy Technology Data Exchange (ETDEWEB)

    Lockwood, Steven John; Voigt, James A.; Tuttle, Bruce Andrew; Bell, Nelson Simmons

    2005-01-01

    Chemically prepared zinc oxide powders are fabricated for the production of high aspect ratio varistor components. Colloidal processing in water was performed to reduce agglomerates to primary particles, form a high solids loading slurry, and prevent dopant migration. The milled and dispersed powder exhibited a viscoelastic to elastic behavioral transition at a volume loading of 43-46%. The origin of this transition was studied using acoustic spectroscopy, zeta potential measurements and oscillatory rheology. The phenomenon occurs due to a volume fraction solids dependent reduction in the zeta potential of the solid phase. It is postulated to result from divalent ion binding within the polyelectrolyte dispersant chain, and was mitigated using a polyethylene glycol plasticizing additive. Chemically prepared zinc oxide powders were processed for the production of high aspect ratio varistor components. Near net shape casting methods including slip casting and agarose gelcasting were evaluated for effectiveness in achieving a uniform green microstructure achieving density values near the theoretical maximum during sintering. The structure of the green parts was examined by mercury porisimetry. Agarose gelcasting produced green parts with low solids loading values and did not achieve high fired density. Isopressing the agarose cast parts after drying raised the fired density to greater than 95%, but the parts exhibited catastrophic shorting during electrical testing. Slip casting produced high green density parts, which exhibited high fired density values. The electrical characteristics of slip cast parts are comparable with dry pressed powder compacts. Alternative methods for near net shape forming of ceramic dispersions were investigated for use with the chemically prepared ZnO material. Recommendations for further investigation to achieve a viable production process are presented.

  10. Preparation of open porous polycaprolactone microspheres and their applications as effective cell carriers in hydrogel system

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Qingchun [Key Laboratory for Ultrafine Materials of Ministry of Education, School of Materials Science and Engineering (China); Tan, Ke; Ye, Zhaoyang [State Key Laboratory of Bioreactor Engineering, School of Bioengineering, East China University of Science and Technology, Shanghai, 200237 China (China); Zhang, Yan, E-mail: zhang_yan@ecust.edu.cn [Key Laboratory for Ultrafine Materials of Ministry of Education, School of Materials Science and Engineering (China); Tan, Wensong [State Key Laboratory of Bioreactor Engineering, School of Bioengineering, East China University of Science and Technology, Shanghai, 200237 China (China); Lang, Meidong, E-mail: mdlang@ecust.edu.cn [Key Laboratory for Ultrafine Materials of Ministry of Education, School of Materials Science and Engineering (China)

    2012-12-01

    Common hydrogel, composed of synthetic polymers or natural polysaccharides could not support the adhesion of anchorage-dependent cells due to the lack of cell affinitive interface and high cell constraint. The use of porous polyester microspheres as cell-carriers and introduction of cell-loaded microspheres into the hydrogel system might overcome the problem. However, the preparation of the open porous microsphere especially using polycaprolactone (PCL) has been rarely reported. Here, the open porous PCL microspheres were fabricated via the combined emulsion/solvent evaporation and particle leaching method. The microspheres exhibited porous surface and inter-connective pore structure. Additionally, the pore structure could be easily controlled by adjusting the processing parameters. The surface pore size could be altered from 20 {mu}m to 80 {mu}m and the internal porosities were varied from 30% to 70%. The obtained microspheres were evaluated to delivery mesenchymal stem cells (MSCs) and showed the improved cell adhesion and growth when compared with the non-porous microspheres. Then, the MSCs loaded microspheres were introduced into agarose hydrogel. MSCs remained alive and sustained proliferation in microsphere/agarose composite in 5-day incubation while a decrement of MSCs viabilities was found in agarose hydrogel without microspheres. The results indicated that the microsphere/hydrogel composite had a great potential in cell therapy and injectable system for tissue regeneration. Highlights: Black-Right-Pointing-Pointer The open porous polycaprolactone microspheres were fabricated using paraffin as a porogen. Black-Right-Pointing-Pointer The microspheres exhibited porous surface and inter-connective pore structure. Black-Right-Pointing-Pointer The surface and internal pore size and porosity of microsphere could be controlled. Black-Right-Pointing-Pointer The porous microspheres exhibited an improved cell adhesion and proliferation. Black

  11. Fibronectin- and collagen-mimetic ligands regulate bone marrow stromal cell chondrogenesis in three-dimensional hydrogels

    Directory of Open Access Journals (Sweden)

    JT Connelly

    2011-09-01

    Full Text Available Modification of tissue engineering scaffolds with bioactive molecules is a potential strategy for modulating cell behavior and guiding tissue regeneration. While adhesion to RGD peptides has been shown to inhibit in vitro chondrogenesis, the effects of extracellular matrix (ECM-mimetic ligands with complex secondary and tertiary structures are unknown. This study aimed to determine whether collagen- and fibronectin-mimetic ligands would retain biologic functionality in three-dimensional (3D hydrogels, whether different ECM-mimetic ligands differentially influence in vitro chondrogenesis, and if effects of ligands on differentiation depend on soluble biochemical stimuli. A linear RGD peptide, a recombinant fibronectin fragment containing the seven to ten Type III repeats (FnIII7-10 and a triple helical, collagen mimetic peptide with the GFOGER motif were covalently coupled to agarose gels using the sulfo-SANPAH crosslinker, and bone marrow stromal cells (BMSCs were cultured within the 3D hydrogels. The ligands retained biologic functionality within the agarose gels and promoted density-dependent BMSC spreading. Interactions with all adhesive ligands inhibited stimulation by chondrogenic factors of collagen Type II and aggrecan mRNA levels and deposition of sulfated glycosaminoglycans. In medium containing fetal bovine serum, interactions with the GFOGER peptide enhanced mRNA expression of the osteogenic gene osteocalcin whereas FnIII7-10 inhibited osteocalcin expression. In conclusion, modification of agarose hydrogels with ECM-mimetic ligands can influence the differentiation of BMSCs in a manner that depends strongly on the presence and nature of soluble biochemical stimuli.

  12. Protocol: a rapid and economical procedure for purification of plasmid or plant DNA with diverse applications in plant biology

    Directory of Open Access Journals (Sweden)

    Li Li

    2010-01-01

    Full Text Available Abstract Research in plant molecular biology involves DNA purification on a daily basis. Although different commercial kits enable convenient extraction of high-quality DNA from E. coli cells, PCR and agarose gel samples as well as plant tissues, each kit is designed for a particular type of DNA extraction work, and the cost of purchasing these kits over a long run can be considerable. Furthermore, a simple method for the isolation of binary plasmid from Agrobacterium tumefaciens cells with satisfactory yield is lacking. Here we describe an easy protocol using homemade silicon dioxide matrix and seven simple solutions for DNA extraction from E. coli and A. tumefaciens cells, PCR and restriction digests, agarose gel slices, and plant tissues. Compared with the commercial kits, this protocol allows rapid DNA purification from diverse sources with comparable yield and purity at negligible cost. Following this protocol, we have demonstrated: (1 DNA fragments as small as a MYC-epitope tag coding sequence can be successfully recovered from an agarose gel slice; (2 Miniprep DNA from E. coli can be eluted with as little as 5 μl water, leading to high DNA concentrations (>1 μg/μl for efficient biolistic bombardment of Arabidopsis seedlings, polyethylene glycol (PEG-mediated Arabidopsis protoplast transfection and maize protoplast electroporation; (3 Binary plasmid DNA prepared from A. tumefaciens is suitable for verification by restriction analysis without the need for large scale propagation; (4 High-quality genomic DNA is readily isolated from several plant species including Arabidopsis, tobacco and maize. Thus, the silicon dioxide matrix-based DNA purification protocol offers an easy, efficient and economical way to extract DNA for various purposes in plant research.

  13. Radiation effects on human glia and glioma cells in vitro

    International Nuclear Information System (INIS)

    The radiosensitivity of human glia and glioma cells has been studied in vitro, and a new cloning method has been developed to overcome the difficulties due to the very low cloning efficiency of these cells. The cells were confined to small palladium areas surrounded by agarose, which increased the cell density, but kept the clones separated. Using this method, the glia cells were found to be very sensitive to gamma irradiation (D0=1.0-1.5 Gy and n=1) in comparision with the glioma cells (D0=1.5-2.5 Gy and n=3.5). The induction and repair of DNA strand breaks were studied with two DNA unwinding techniques. No differences between the two cell-lines were detected when induction and fast repair were studied with the single-labelling method, while the glioma cells showed less unrepaired DNA strand breaks than the glia cells after 1, 2 and 3 hours, when the double-labelling method was used. Detachment, attachment and growth kinetics were studied using the palladium-agarose cloning method. All of the glioma cell-lines studied, detached and attached themselves at rates higher than the normal diploid glia cell-lines. All of the cell-lines contained clones with different properties. Some clones were rapidly growing, others maintained a nearly constant number of cells or even decreased. The effects of chronic hypoxia were tested in a few experiments. Low oxygen tension in the culture medium reduced the rate of growth and the DNA synthesis of the glioma cells. The present study indicates that cultured human glioma cells are less radiosensitive than cultured glia cells. The palladium-agarose technique, enable studying growth kinetics detachment, attachment and radiosensitivity in a quantitative manner for cells with low cloning efficiency. (author)

  14. Local Delivery of Fluorescent Dye For Fiber-Optics Confocal Microscopy of the Living Heart

    Directory of Open Access Journals (Sweden)

    Chao eHuang

    2014-09-01

    Full Text Available Fiber-optics confocal microscopy (FCM is an emerging imaging technology with various applications in basic research and clinical diagnosis. FCM allows for real-time in situ microscopy of tissue at sub-cellular scale. Recently FCM has been investigated for cardiac imaging, in particular, for discrimination of cardiac tissue during pediatric open-heart surgery. FCM relies on fluorescent dyes. The current clinical approach of dye delivery is based on systemic injection, which is associated with high dye consumption and adverse clinical events. In this study, we investigated approaches for local dye delivery during FCM imaging based on dye carriers attached to the imaging probe. Using three-dimensional confocal microscopy, automated bench tests, and FCM imaging we quantitatively characterized dye release of carriers composed of open-pore foam only and foam loaded with agarose hydrogel. In addition, we compared local dye delivery with a model of systemic dye delivery in the isolated perfused rodent heart. We measured the signal-to-noise ratio of images acquired in various regions of the heart. Our evaluations showed that foam-agarose dye carriers exhibited a prolonged dye release versus foam-only carriers. Foam-agarose dye carriers allowed reliable imaging of 5-9 lines, which is comparable to 4-8 min of continuous dye release. Our study in the living heart revealed that the SNR of FCM images using local and systemic dye delivery is not different. However, we observed differences in the imaged tissue microstructure with the two approaches. Structural features characteristic of microvasculature were solely observed for systemic dye delivery. Our findings suggest that local dye delivery approach for FCM imaging constitutes an important alternative to systemic dye delivery. We suggest that the approach for local dye delivery will facilitate clinical translation of FCM, for instance, for FCM imaging during pediatric heart surgery.

  15. Apolipoprotein A-I metabolism in cynomolgus monkey. Identification and characterization of beta-migrating pools

    International Nuclear Information System (INIS)

    Fresh plasma from control (C) and hypercholesterolemic (HC) cynomolgus monkeys was analyzed by agarose electrophoresis-immunoblotting with antibody to cynomolgus monkey apolipoprotein (apo) A-I. Two bands were evident on the autoradiogram: an alpha-migrating band (high density lipoprotein) and a beta-migrating band that comigrated exactly with cynomolgus monkey low density lipoprotein (LDL). The presence of beta-migrating apo A-I in the plasma of these monkeys was confirmed by Geon-Pevikon preparative electrophoresis, crossed immunoelectrophoresis, and isotope dilution studies in which radiolabeled apo A-I was found to equilibrate also with alpha- and beta-migrating pools of apo A-I in the plasma. Subfractionation of C and HC plasma by agarose column chromatography (Bio-Gel A-0.5M and A-15M) followed by agarose electrophoresis-immunoblotting indicated that the beta-migrating apo A-I in C was relatively homogeneous and eluted with proteins of Mr approximately 50 kD [apo A-I(50 kD)], whereas two beta-migrating fractions were identified in HC, one that eluted with the 50-kD proteins, and the other that eluted in the LDL Mr range [apo A-I(LDL)]. The apo A-I(LDL) was precipitated by antibody to cynomolgus monkey apo B. The apo A-I(50 kD) accounted for 5 +/- 1% (mean +/- SD) of the plasma apo A-I in C plasma, and 15 +/- 7% in HC plasma. No apo A-I(LDL) was detected in C plasma, but that fraction accounted for 9 +/- 7% of the apo A-I in HC plasma. These data establish the presence of multiple pools of apo A-I in the cynomolgus monkey, which must be taken into consideration in any comprehensive model of apo A-I metabolism in this species

  16. Novel ATP-binding heat-inducible protein of Mr = 37,000 that is sensitive to transformation in BALB/3T3 cells

    International Nuclear Information System (INIS)

    Using affinity chromatography on ATP-agarose, we have identified a major ATP-binding protein in Nonidet P-40 extracts of avian and mammalian cells labeled with [35S]methionine. After washing ATP-agarose beads with high-ionic-strength buffer (0.4 M NaCl), the 37-kD protein was shown to be one of the major ATP-binding proteins while p72 and grp78, which are members of the hsp70 family, also bound to ATP-agarose. This protein consisted of several spots on two-dimensional gel electrophoresis. The isoelectric point of the most basic spot was approximately 9.2 in chick embryo fibroblasts, whereas it was about 8.8 in mouse 3T3 cells. The identities of these proteins in mouse and chick cells were confirmed by peptide mapping. After heat-shock treatment of BALB/3T3 cells, the major heat-shock protein, hsp70, was shown to be induced very rapidly after heat shock and was recovered in the ATP-binding fraction. Besides hsp70, a 37-kD protein was also found to be induced by heat shock. This protein was drastically induced by treating the cells with alpha,alpha'-dipyridyl, an iron chelating reagent, but not with sodium arsenite, calcium ionophore, or tunicamycin. The synthesis and the total amount of this ATP-binding protein increased in mouse 3T3 cells transformed by simian virus 40, methylcholanthrene, or activated c-Ha-ras oncogene compared to their normal counterparts. The incorporation of [32P]orthophosphate was not detected in either normal or transformed cells. These studies established that a major ATP-binding protein of Mr = 37,000 is a heat-inducible protein and that the synthesis of this protein is regulated by malignant transformation

  17. Preparation of open porous polycaprolactone microspheres and their applications as effective cell carriers in hydrogel system

    International Nuclear Information System (INIS)

    Common hydrogel, composed of synthetic polymers or natural polysaccharides could not support the adhesion of anchorage-dependent cells due to the lack of cell affinitive interface and high cell constraint. The use of porous polyester microspheres as cell-carriers and introduction of cell-loaded microspheres into the hydrogel system might overcome the problem. However, the preparation of the open porous microsphere especially using polycaprolactone (PCL) has been rarely reported. Here, the open porous PCL microspheres were fabricated via the combined emulsion/solvent evaporation and particle leaching method. The microspheres exhibited porous surface and inter-connective pore structure. Additionally, the pore structure could be easily controlled by adjusting the processing parameters. The surface pore size could be altered from 20 μm to 80 μm and the internal porosities were varied from 30% to 70%. The obtained microspheres were evaluated to delivery mesenchymal stem cells (MSCs) and showed the improved cell adhesion and growth when compared with the non-porous microspheres. Then, the MSCs loaded microspheres were introduced into agarose hydrogel. MSCs remained alive and sustained proliferation in microsphere/agarose composite in 5-day incubation while a decrement of MSCs viabilities was found in agarose hydrogel without microspheres. The results indicated that the microsphere/hydrogel composite had a great potential in cell therapy and injectable system for tissue regeneration. Highlights: ► The open porous polycaprolactone microspheres were fabricated using paraffin as a porogen. ► The microspheres exhibited porous surface and inter-connective pore structure. ► The surface and internal pore size and porosity of microsphere could be controlled. ► The porous microspheres exhibited an improved cell adhesion and proliferation. ► Mesenchymal stem cells survived and proliferated in microsphere/hydrogel composite.

  18. Effective chemotherapy induce apoptosis in vivo in patients with leukemia

    Institute of Scientific and Technical Information of China (English)

    岑溪南; 朱平; 虞积仁; 石永进; 马明信

    2003-01-01

    Objective To investigate apoptosis in vivo in patients with leukemia at different stages of the first cycle of chemotherapy.Methods We detected apoptosis of HL-60 cells and peripheral blood leukemia cells in 17 patients at different stages, using in situ terminal deoxynucleotidyl transferase (TdT) fluorescence measurement and DNA electrophoresis. Results When HL-60 cells were incubated with 0.02 mg/L harringtonine for 0 to 48 hours, agarose gel electrophoresis showed that DNA ladder patterns became evident only at 12 hour into the treatment. In situ TdT assay showed that apoptotic cells occurred after one hour of the treatment. Apoptotic cells were few (0-3.3%) before chemotherapy, but increased substantially (11.4%-87.5%) during chemotherapy in patients with complete remission (CR) or partial remission (PR). Apoptotic cells were few (0-6.1%) during chemotherapy in ten patients with no remission (NR). DNA ladder cannot be detected by agarose gel electrophoresis either before, during or after chemotherapy. Wilcoxon signed rank test shows: P=0.0012<0.01, apoptotic cells during chemotherapy were present in greater quantity than prior to chemotherapy. Wilcoxon rank sum test shows: P=0.0011<0.01, with the median of apoptotic cells during chemotherapy in patients with CR or PR more than with NR.Conclusions TdT assay can be used to detect apoptotic cells earlier and more sensitively than DNA agarose gel electrophoresis. In situ TdT assay is useful to detect apoptosis in vivo in the initial phase of chemotherapy for immediate modification of the chemotherapy regimen, whereas electrophoretic analysis is not sensitive enough to detect apoptotic cell in vivo. Where the median of apoptotic cells during chemotherapy in patients with CR or PR were greater than with NR, only effective drug therapy could induce apoptosis.

  19. Insight into electrochemical properties of Co3O4–modified magnetic polymer electrolyte

    International Nuclear Information System (INIS)

    Highlights: • A novel cobaltosic oxide-modified magnetic agarose electrolyte. • Magnetic field–induced ordered microstructure and increased ionic conductivity. • Improved recombination process and good long-term stability of DSSCs after magnetic field treatment. • Better photovoltaic performance of the Co3O4-modified DSSC than that of NiO-modified DSSC under magnetic field treatment. - Abstract: Agarose–based electrolyte containing magnetic Co3O4 nanoparticles is studied for quasi–solid–state dye–sensitized solar cells (DSSCs) under external magnetic field treatment. SEM studies reveal the existence of oriented microstructure in Co3O4–modified agarose electrolyte film under proper magnetic field intensity. The formation mechanism of this ordered structure induced by magnetic field is analyzed. The impedance analysis shows that the ionic conductivity of Co3O4–modified agarose electrolyte is obviously increased by applying magnetic field intensity of 25 mT. Improved electron recombination process and photoelectric performance are observed in DSSCs under certain magnetic field treatment by electrochemical impedance spectra (EIS) and photovoltaic studies. The DSSC treated with magnetic field can maintain the efficiency unchanged for 434 hours without sealing. This is attributed to the high ionic conductivity and improved electron transfer process in DSSC resulting from the magnetic field treatment. Comparison of photovoltaic performances for Co3O4 and NiO modified DSSCs under 25 mT magnetic field treatment shows that Co3O4-modified DSSC exhibits higher energy conversion efficiency than that of NiO-modified one at the same condition

  20. Experimental Assays to Assess the Efficacy of Vinegar and Other Topical First-Aid Approaches on Cubozoan (Alatina alata Tentacle Firing and Venom Toxicity

    Directory of Open Access Journals (Sweden)

    Angel A. Yanagihara

    2016-01-01

    Full Text Available Despite the medical urgency presented by cubozoan envenomations, ineffective and contradictory first-aid management recommendations persist. A critical barrier to progress has been the lack of readily available and reproducible envenomation assays that (1 recapitulate live-tentacle stings; (2 allow quantitation and imaging of cnidae discharge; (3 allow primary quantitation of venom toxicity; and (4 employ rigorous controls. We report the implementation of an integrated array of three experimental approaches designed to meet the above-stated criteria. Mechanistically overlapping, yet distinct, the three approaches comprised (1 direct application of test solutions on live tentacles (termed tentacle solution assay, or TSA with single image- and video-microscopy; (2 spontaneous stinging assay using freshly excised tentacles overlaid on substrate of live human red blood cells suspended in agarose (tentacle blood agarose assays, or TBAA; and (3 a “skin” covered adaptation of TBAA (tentacle skin blood agarose assay, or TSBAA. We report the use and results of these assays to evaluate the efficacy of topical first-aid approaches to inhibit tentacle firing and venom activity. TSA results included the potent stimulation of massive cnidae discharge by alcohols but only moderate induction by urine, freshwater, and “cola” (carbonated soft drink. Although vinegar, the 40-year field standard of first aid for the removal of adherent tentacles, completely inhibited cnidae firing in TSA and TSBAA ex vivo models, the most striking inhibition of both tentacle firing and subsequent venom-induced hemolysis was observed using newly-developed proprietary formulations (Sting No More™ containing copper gluconate, magnesium sulfate, and urea.

  1. Preparation of peptide-functionalized gold nanoparticles using one pot EDC/sulfo-NHS coupling.

    Science.gov (United States)

    Bartczak, Dorota; Kanaras, Antonios G

    2011-08-16

    Although carbodiimides and succinimides are broadly employed for the formation of amide bonds (i.e., in amino acid coupling), their use in the coupling of peptides to water-soluble carboxylic-terminated colloidal gold nanoparticles remains challenging. In this article, we present an optimization study for the successful coupling of the KPQPRPLS peptide to spherical and rodlike colloidal gold nanoparticles. We show that the concentration, reaction time, and chemical environment are all critical to achieving the formation of robust, peptide-coated colloidal nanoparticles. Agarose gel electrophoresis was used for the characterization of conjugates. PMID:21728291

  2. Using restriction mapping to teach basic skills in the molecular biology lab.

    Science.gov (United States)

    Walsh, Lauren; Shaker, Elizabeth; De Stasio, Elizabeth A

    2007-05-01

    Digestion of DNA with restriction enzymes, calculation of volumes and concentrations of reagents for reactions, and the separation of DNA fragments by agarose gel electrophoresis are common molecular biology techniques that are best taught through repetition. The following open-ended, investigative laboratory exercise in plasmid restriction mapping allows students to gain technical expertise while simultaneously exploring the utility of gel electrophoresis and restriction mapping. Because of its interpretive nature, this project also provides data suitable for a written report, and can thus be used to reinforce lessons on figure presentation and science writing skills. PMID:21591089

  3. Genotoxic and Cytotoxic Safety Evaluation of Papain (Carica papaya L. Using In Vitro Assays

    Directory of Open Access Journals (Sweden)

    Claudia R. da Silva

    2010-01-01

    This work evaluated the toxic and mutagenic potential of papain and its potential antioxidant activity against induced-H2O2 oxidative stress in Escherichia coli strains. Cytotoxicity assay, Growth inhibition test, WP2-Mutoxitest and Plasmid-DNA treatment, and agarose gel electrophoresis were used to investigate if papain would present any toxic or mutagenic potential as well as if papain would display antioxidant properties. Papain exhibited negative results for all tests. This agent presented an activity protecting cells against H2O2-induced mutagenesis.

  4. Isolation and partial characterization of a D-galactose-binding lectin from the latex of Synadenium carinatum

    OpenAIRE

    Maria Aparecida de Souza; Francielle Amâncio-Pereira; Cristina Ribeiro de Barros Cardoso; Adriano Gomes da Silva; Edmar Gomes Silva; Lívia Resende Andrade; Janethe Deolina Oliveira Pena; Henrique Lanza; Sandra Regina Afonso-Cardoso

    2005-01-01

    A lectin from the latex of Synadenium carinatum was purified by affinity chromatography on immobilized-D-galactose-agarose and shown to be a potent agglutinin of human erythrocytes. The haemagglutination of human red cells was inhibited by 3.0 mM N-acetyl-D-galactopyranoside, 6.3 mM methyl-beta-D-galactopyranoside, 50 mM methyl-alpha-D-galactopyranoside and 50 mM D-fucose but not by L-fucose, demonstrating an anomeric and a conformational specificity. According to SDS-PAGE analysis, the lecti...

  5. Dois novos sistemas de diagnose precoce da meleira do mamoeiro Two new systems of early diagnosis of papaya sticky disease

    OpenAIRE

    Eder T. Tavares; JOSELI S. TATAGIBA; José A. Ventura; Manoel T. Souza Jr.

    2004-01-01

    A diagnose da meleira do mamoeiro (Carica papaya)tem sido feita mediante observação de sintomas que aparecem principalmente nos frutos ou pela detecção de dsRNA de cerca de 12 kb, purificado a partir do látex em coluna de CF11, em gel de agarose ou poliacrilamida. Os sintomas nos frutos são tardios e permitem longa permanência de plantas infectadas no campo e o processo usado para detectar dsRNA é laborioso, não se prestando a detecção em larga escala. Visando disponibilizar protocolos de dia...

  6. A chemically cleavable biotinylated nucleotide: usefulness in the recovery of protein-DNA complexes from avidin affinity columns.

    OpenAIRE

    Shimkus, M; Levy, J; Herman, T

    1985-01-01

    A biotinylated nucleotide analog containing a disulfide bond in the 12-atom linker joining biotin to the C-5 of the pyrimidine ring has been synthesized. This analog, Bio-SS-dUTP, is an efficient substrate for Escherichia coli DNA polymerase I. Bio-SS-dUTP supported DNA synthesis in a standard nick-translation reaction at 35%-40% the rate of an equal concentration of the normal nucleotide, TTP. DNA containing this analog was bound to an avidin-agarose affinity column and subsequently eluted a...

  7. Methylase-limited partial NotI cleavage for physical mapping of genomic DNA.

    OpenAIRE

    Hanish, J; McClelland, M

    1990-01-01

    Partial cleavage of DNA with the restriction endonuclease NotI (5'...GC/GGCCGC...3') is an important technique for genomic mapping. However, partial genomic cleavage with this enzyme is impaired by the agarose matrix in which the DNA must be suspended. To solve this problem we have purified the blocking methylase M. BspRI (5'...GGmCC...3') for competition digests with NotI. The resulting methylase-limited partial DNA cleavage is shown to be superior to standard techniques on bacterial genomic...

  8. Sequence-specific double-strand cleavage of DNA by penta-N-methylpyrrolecarboxamide-EDTA·Fe(II)

    OpenAIRE

    Schultz, Peter G.; Dervan, Peter B.

    1983-01-01

    In the presence of O2 and 5 mM dithiothreitol, penta-N-methylpyrrolecarboxamide-EDTA·Fe(II) [P5E·Fe(II)] at 0.5 µ M cleaves pBR322 plasmid DNA (50 µ M in base pairs) on opposite strands to afford discrete DNA fragments as analyzed by agarose gel electrophoresis. High-resolution denaturing gel electrophoresis of a 32P-end-labeled 517-base-pair restriction fragment containing a major cleavage site reveals that P5E·Fe(II) cleaves 3-5 base pairs contiguous to a 6-base-pair sequence, 5'-T-T-T-T-T-...

  9. Sequence-specific double-strand cleavage of DNA by penta-N-methylpyrrolecarboxamide-EDTA X Fe(II).

    OpenAIRE

    Schultz, P G; Dervan, P B

    1983-01-01

    In the presence of O2 and 5 mM dithiothreitol, penta-N-methylpyrrolecarboxamide-EDTA X Fe(II) [P5E X Fe(II)] at 0.5 microM cleaves pBR322 plasmid DNA (50 microM in base pairs) on opposite strands to afford discrete DNA fragments as analyzed by agarose gel electrophoresis. High-resolution denaturing gel electrophoresis of a 32P-end-labeled 517-base-pair restriction fragment containing a major cleavage site reveals that P5E X Fe(II) cleaves 3-5 base pairs contiguous to a 6-base-pair sequence, 5...

  10. Soroprevalência de brucelose canina na cidade de Alfenas, MG: dados preliminares

    Directory of Open Access Journals (Sweden)

    Almeida A.C.

    2001-01-01

    Full Text Available One hundred and two blood serum samples from dogs referred to the Veterinary Teaching Hospital of Alfenas University, were submitted to the agarose immune diffusion and to the fast serum-agglutination tests addressed to find antibodies anti- Brucella canis and B. abortus, respectively. Five samples (4.9% were positives for B. canis and none for B. abortus. This prevalence is considered by some as an alert for an epidemiologic and public healthy problems, considering the serious zoonotic aspect of canine brucellosis

  11. Rapid isolation of both double-stranded RNA and PCR-suitable DNA from the obligate biotrophic phytopathogenic fungus Uncinula necator using a commercially available reagent.

    Science.gov (United States)

    Délye, C; Corio-Costet, M F

    1998-10-01

    A method for rapid extraction of both double-stranded RNA (dsRNA) and DNA from an obligate biotrophic phytopathogenic fungus is described. Lyophilised fungal material is incubated in a commercial guanidium thiocyanate reagent. Proteins and cell debris are centrifuged by chloroform precipitation. After precipitation in isopropanol and washing in 75% ethanol, nucleic acids are resuspended in water (10 microl/mg fungal dry weight). DsRNA is directly visualised by agarose gel electrophoresis. DNA contained in 10-fold dilutions of the samples proved to be suitable for PCR-based experiments. PMID:9779614

  12. Detection of hepatitis C virus RNA using reverse transcription PCR

    International Nuclear Information System (INIS)

    Detection of the viral genome (HCV RNA) is by a combination of cDNA synthesis and PCR followed by gel analysis and/or hybridization assay. In principle, cDNA is synthesized using the viral RNA as template and the enzyme, reverse transcriptase. The cDNA is then amplified by PCR and the product detected. Agarose gel electrophoresis provides a rapid and simple detection method; however, it is non-quantitative. The assay protocol described in this paper is adapted from that published by Chan et al. Comments on various aspects of the assay are based on experience with the method in our laboratory

  13. Human complement component C3

    DEFF Research Database (Denmark)

    Behrendt, N

    1985-01-01

    The two common genetic variants of human C3, C3 S and C3 F, were purified and characterized by SDS-PAGE, agarose gel electrophoresis, isoelectric focusing and amino acid analysis. The difference in electrophoretic mobility between the two variants was conserved after purification, and by...... isoelectric focusing of the hemolytically active proteins, pI values of 5.86 and 5.81 were determined for C3 S and C3 F, respectively. Any difference in amino acid composition was too small to be detected by amino acid analysis, and the two proteins had the same molecular weight as determined by SDS-PAGE....

  14. Low-Molecular Weight Polyethylenimine Modified with Pluronic 123 and RGD- or Chimeric RGD-NLS Peptide: Characteristics and Transfection Efficacy of Their Complexes with Plasmid DNA

    OpenAIRE

    Jing Hu; Wenfang Zhao; Kehai Liu; Qian Yu; Yuan Mao; Zeyu Lu; Yaguang Zhang; Manman Zhu

    2016-01-01

    To solve the problem of transfection efficiency vs. cytotoxicity and tumor-targeting ability when polyethylenimine (PEI) was used as a nonviral gene delivery vector, new degradable PEI polymers were synthesized via cross-linking low-molecular-weight PEI with Pluronic P123 and then further coupled with a targeting peptide R4 (RGD) and a bifunctional R11 (RGD-NLS), which were termed as P123-PEI-R4 and P123-PEI-R11, respectively. Agarose gel electrophoresis showed that both P123-PEI-R4 and P123-...

  15. Interference of humic acids and DNA extracted directly from soil in detection and transformation of recombinant DNA from bacteria and a yeast.

    OpenAIRE

    Tebbe, C C; Vahjen, W.

    1993-01-01

    A two-step protocol for the extraction and purification of total DNA from soil samples was developed. Crude DNA extracts (100 microliters from 5 g of soil) were contaminated with humic acids at concentrations of 0.7 to 3.3 micrograms/microliters, depending on the type of soil extracted. The coextracted humic acid fraction of a clay silt was similar to a commercially available standard humic acid mixture, as determined by electrophoretic mobility in agarose gels, UV fluorescence, and inhibitio...

  16. Neutral Comet Assay

    OpenAIRE

    2013-01-01

    The Comet assay (or Single Cell Gel Electrophoresis assay) is a sensitive technique to detect DNA damage at the level of an individual cell. This technique is based on micro-electrophoresis of cells DNA content. Briefly, cells are embedded in agarose, lysed and submitted to an electric field, before the staining step with a fluorescent DNA binding dye. Damaged DNA (charged DNA) migrates in this field, forming the tail of a “comet”, while undamaged DNA remained in the head of the “comet”. The ...

  17. Identification of a cAMP-dependent protein kinase in bovine and human follicular fluids.

    Science.gov (United States)

    Yang, L S; Kadam, A L; Koide, S S

    1993-11-01

    A soluble protein kinase (PK) was purified from bovine and human follicular fluids (FF) by ultrafiltration through a PM-10 membrane followed by chromatography on heparin-agarose, DEAE-cellulose and cellulose phosphate columns. The PK phosphorylated calf thymus histones and endogenous FF proteins having estimated Mrs of 40, 62, 128 and 180 KD. cAMP enhanced PK activity; whereas protein kinase A (PKA)-inhibitor peptide blocked the activity. The present findings suggest that the enzyme is a cAMP-dependent PK. PMID:8118427

  18. Four cleaved amplified polymorphic sequence (CAPS) markers for the detection of the Juglans ailantifolia chloroplast in putatively native J. cinerea populations.

    Science.gov (United States)

    McCleary, Tim S; Robichaud, Rodney L; Nuanes, Steve; Anagnostakis, Sandra L; Schlarbaum, Scott E; Romero-Severson, Jeanne

    2009-03-01

    Hybridization between butternut (Juglans cinerea), a forest tree native to eastern North America, and Japanese walnut (J. ailantifolia), a tree tolerant to the lethal fungal disease butternut canker, casts doubt on the genetic identity of the remaining butternuts. We report a diagnostic test to distinguish the J. cinerea chloroplast from the J. ailantifolia chloroplast using cleaved amplified polymorphic sequences resolvable in 1.5% agarose gels. J. ailantifolia maternal ancestry in naturally regenerated stands provides a site selection criterion for studies of introgression dynamics when the non-native parent and the hybrids tolerate a disease to which the native species is susceptible. PMID:21564682

  19. Multichannel microscale system for high throughput preparative separation with comprehensive collection and analysis

    Energy Technology Data Exchange (ETDEWEB)

    Karger, Barry L.; Kotler, Lev; Foret, Frantisek; Minarik, Marek; Kleparnik, Karel

    2003-12-09

    A modular multiple lane or capillary electrophoresis (chromatography) system that permits automated parallel separation and comprehensive collection of all fractions from samples in all lanes or columns, with the option of further on-line automated sample fraction analysis, is disclosed. Preferably, fractions are collected in a multi-well fraction collection unit, or plate (40). The multi-well collection plate (40) is preferably made of a solvent permeable gel, most preferably a hydrophilic, polymeric gel such as agarose or cross-linked polyacrylamide.

  20. Identification of cAMP-dependent phosphorylated proteins involved in the formation of environment-resistant resting cysts by the terrestrial ciliate Colpoda cucullus

    Directory of Open Access Journals (Sweden)

    Y Sogame

    2014-07-01

    Full Text Available In the terrestrial ciliate Colpoda cucullus, an elevation of the intracellular cAMP concentration was reported to be involved in environment-resistant resting cyst formation. In the present study, cAMP-dependently phosphorylated proteins of encystment-induced C. cucullus were isolated with Phos-tag agarose phosphate-affinity beads and subsequent SDS-PAGE. In a liquid chromatography/tandem mass spectrometry analysis of these phosphoproteins, 27-, 37- and 43-kDa proteins (p27, p37 and p43 were identified as Rieske iron-sulfur protein, histone H4 (hyperacetylated form, and actin, respectively.

  1. Identification of a novel 70 kDa protein that binds to the core promoter element and is essential for ribosomal DNA transcription

    OpenAIRE

    Yamamoto, Kazuo; Koga, Atsuro; Yamamoto, Mika; Nishi, Yu-ichi; Tamura, Taka-aki; Nogi, Yasuhisa; Muramatsu, Masami

    2000-01-01

    Mammalian ribosomal RNA genes (rDNA) are transcribed by RNA polymerase I and at least two auxiliary factors, UBF and SL1/TFID/TIF-IB. It has also been reported that an additional factor(s) is required to reconstitute efficient initiation of rDNA transcription in vitro, depending upon the procedures of chromatographic separation. In an attempt to elucidate the molecular identity of such yet uncertain activities, we have developed agarose gel shift and UV cross-linking assays to detect proteins...

  2. Alkaline gel electrophoresis assay to detect DNA strand breaks and repair mechanisms in Escherichia coli

    Directory of Open Access Journals (Sweden)

    José Carlos Pelielo de Mattos

    2008-12-01

    Full Text Available Reactive oxygen species (ROS can induce lesions in different cellular targets, including DNA. Stannous chloride (SnCl2 is a ROS generator, leading to lethality in Escherichia coli (E. coli, with the base excision repair (BER mechanism playing a role in this process. Many techniques have been developed to detect genotoxicity, as comet assay, in eukaryotic cells, and plasmid DNA agarose gel electrophoresis. In this study, an adaptation of the alkaline gel electrophoresis method was carried out to ascertain the induction of strand breaks by SnCl2 in bacterial DNA, from E. coli BER mutants, and its repair pathway. Results obtained show that SnCl2 was able to induce DNA strand breaks in all strains tested. Moreover, endonuclease IV and exonuclease III play a role in DNA repair. On the whole, data has shown that the alkaline gel electrophoresis assay could be used both for studying DNA strand breaks induction and for associated repair mechanisms.Espécies reativas de oxigênio (ERO podem induzir lesões em diferentes alvos celulares, incluindo o DNA. O cloreto estanoso (SnCl2 é um gerador de ERO que induz letalidade em E. coli, sendo o reparo por excisão de bases (BER um mecanismo importante neste processo. Técnicas como o ensaio cometa (em eucariotos e a eletroforese de DNA plasmidial em gel de agarose têm sido utilizadas para detectar genotoxicidade. No presente estudo, uma adaptação do método de eletroforese em gel alcalino de agarose foi usada para verificar a indução de quebras, pelo SnCl2, no DNA de E. coli, bem como a participação de enzimas do BER na restauração das lesões. Os resultados mostraram que o SnCl2 induziu quebras no DNA de todas as cepas testadas. Além disso, endonuclease IV e exonuclease III estão envolvidas na reparação dos danos. Em resumo, os dados obtidos indicam que a metodologia de eletroforese em gel alcalino de agarose pode ser empregada tanto para o estudo de quebras no DNA, quanto para avaliação dos

  3. Investigation of the repair of single-strand breaks in human DNA using alkaline gel electrophoresis

    International Nuclear Information System (INIS)

    Unstimulated lymphocytes from eight healthy persons were exposed to 10-, 30-, and 100-Gy doses of 60Co gamma radiation. The repair of damaged DNA was measured by (1) alkaline gel electrophoresis (extracted DNA loaded on 0.25% agarose gel, run at 1 V/cm for 39-44 h) at 0, 1, and 2 h after exposure and (2) incorporation of [3H]thymidine into unstimulated lymphocytes in the presence of 2 mM hydroxyurea 1 and 2 h after exposure. Both methods--alkaline gel electrophoresis and thymidine incorporation--showed that repair was completed within 2 h

  4. Undergraduate physics laboratory: Electrophoresis in chromatography paper

    Science.gov (United States)

    Hyde, Alexander; Batishchev, Oleg

    2015-12-01

    An experiment studying the physical principles of electrophoresis in liquids was developed for an undergraduate laboratory. We have improved upon the standard agarose gel electrophoresis experimental regime with a straightforward and cost-effective procedure, in which drops of widely available black food coloring were separated by electric field into their dye components on strips of chromatography paper soaked in a baking soda/water solution. Terminal velocities of seven student-safe dyes were measured as a function of the electric potential applied along the strips. The molecular mobility was introduced and calculated by analyzing data for a single dye. Sources of systematic and random errors were investigated.

  5. Cloning, Expression and Characterization of a Novel Thermophilic Polygalacturonase from Caldicellulosiruptor bescii DSM 6725

    OpenAIRE

    Yanyan Chen; Dejun Sun; Yulai Zhou; Liping Liu; Weiwei Han; Baisong Zheng; Zhi Wang; Zuoming Zhang

    2014-01-01

    We cloned the gene ACM61449 from anaerobic, thermophilic Caldicellulosiruptor bescii, and expressed it in Escherichia coli origami (DE3). After purification through thermal treatment and Ni-NTA agarose column extraction, we characterized the properties of the recombinant protein (CbPelA). The optimal temperature and pH of the protein were 72 °C and 5.2, respectively. CbPelA demonstrated high thermal-stability, with a half-life of 14 h at 70 °C. CbPelA also showed very high activity for polyga...

  6. Analysis of pectate lyases produced by soft rot bacteria associated with spoilage of vegetables.

    OpenAIRE

    Liao, C.H.

    1989-01-01

    Isoelectric focusing (IEF) profiles of pectate lyases (PLs) produced by five different groups of soft rot bacteria were analyzed by using the combined techniques of thin-layer polyacrylamide gel IEF and agarose-pectate overlay activity staining. Four strains of soft rot Erwinia spp. produced three or more PL isozymes. All of eight Pseudomonas viridiflava strains examined produced one single PL with a pI of 9.7. All 10 of Pseudomonas fluorescens strains produced two PLs; the major one had a pI...

  7. Clay nanotube-biopolymer composite scaffolds for tissue engineering

    Science.gov (United States)

    Naumenko, Ekaterina A.; Guryanov, Ivan D.; Yendluri, Raghuvara; Lvov, Yuri M.; Fakhrullin, Rawil F.

    2016-03-01

    Porous biopolymer hydrogels doped at 3-6 wt% with 50 nm diameter/0.8 μm long natural clay nanotubes were produced without any cross-linkers using the freeze-drying method. The enhancement of mechanical strength (doubled pick load), higher water uptake and thermal properties in chitosan-gelatine-agarose hydrogels doped with halloysite was demonstrated. SEM and AFM imaging has shown the even distribution of nanotubes within the scaffolds. We used enhanced dark-field microscopy to visualise the distribution of halloysite nanotubes in the implantation area. In vitro cell adhesion and proliferation on the nanocomposites occur without changes in viability and cytoskeleton formation. In vivo biocompatibility and biodegradability evaluation in rats has confirmed that the scaffolds promote the formation of novel blood vessels around the implantation sites. The scaffolds show excellent resorption within six weeks after implantation in rats. Neo-vascularization observed in newly formed connective tissue placed near the scaffold allows for the complete restoration of blood flow. These phenomena indicate that the halloysite-doped scaffolds are biocompatible as demonstrated both in vitro and in vivo. The chitosan-gelatine-agarose doped clay nanotube nanocomposite scaffolds fabricated in this work are promising candidates for tissue engineering applications.Porous biopolymer hydrogels doped at 3-6 wt% with 50 nm diameter/0.8 μm long natural clay nanotubes were produced without any cross-linkers using the freeze-drying method. The enhancement of mechanical strength (doubled pick load), higher water uptake and thermal properties in chitosan-gelatine-agarose hydrogels doped with halloysite was demonstrated. SEM and AFM imaging has shown the even distribution of nanotubes within the scaffolds. We used enhanced dark-field microscopy to visualise the distribution of halloysite nanotubes in the implantation area. In vitro cell adhesion and proliferation on the nanocomposites occur

  8. A seven-year storage report of good manufacturing practice-grade naked plasmid DNA: stability, topology, and in vitro/in vivo functional analysis

    OpenAIRE

    Walther, W.; Schmeer, M.; Kobelt, D.; Baier, R.; Harder , A.; Walhorn, V.; Anselmetti, D; Aumann, J; Fichtner, I.; Schleef, M

    2013-01-01

    The great interest of naked plasmid DNA in gene therapy studies is reflected by the fact, that it is currently used in 18% of all gene therapy trials. Therefore, validation of topology and functionality of DNA resulting from its long-term stability is an essential requirement for safe and effective gene transfer. To this aim, we analyzed the stability of GMP-grade pCMV-{beta} reporter plasmid DNA by capillary gel electrophoresis, agarose gel electrophoresis and atomic force microscopy. The pl...

  9. Fractionation and characterization of a yeast mRNA splicing extract.

    OpenAIRE

    S. C. Cheng; Abelson, J

    1986-01-01

    We have fractionated a yeast whole cell extract that can accurately splice synthetic actin and CYH2 pre-mRNAs. Three fractions, designated I, II, and III, have been separated by use of ammonium sulfate fractionation and chromatography on heparin agarose. Each fraction alone has no splicing activity. Fractions I and II allow the first step of the splicing reaction to proceed, giving rise to the splicing intermediates, free exon 1, and intron-exon 2. Addition of fraction III completes the react...

  10. Selective adsorption of phosphoproteins on gel-immobilized ferric chelate

    International Nuclear Information System (INIS)

    Ferric ions are very strongly adsorbed to iminodiacetic acid substituted agarose. This firmly immobilized complex acts as a selective immobilized metal affinity adsorbent for phosphoproteins. Chromatography based on this principle is illustrated by the adsorption-desorption behavior of egg yolk phosvitin before and after dephosphorylation as well as by the change in the chromatographic pattern before and after enzymic phosphorylation of selected histones. The strength of binding is dependent on the phosphate content. The difference is binding before and after phosphorylation of a single amino acid residue is demonstrated. Affinity elution can be accomplished by inclusion in the buffer of (1) phosphoserine or (2) a displacing metal ion such as Mg2+

  11. Determination Of Appropriate Antibiotic In Bacterial Meningitis Of Children Based On MIC

    OpenAIRE

    Noorbakhsh S; SA Siadati; Rimaz S; Mamishi S.; Haghi Ashtiani T

    2005-01-01

    Background: Bacterial meningitis is one of the most serious infections in infants and children. Three organisms include S.Pneumo;N.mening;H.Influ are the most common cause of meningitis in children between 2M-14y age.Etest is a new method for determination the MIC of some antimicrobial drugs in agarose .This method is useful for some organisms like as S .Pneumo; N.mening; H.Influ;sensitive Streptococcus and anaerobic ;aerobic gram negative. Materials and Methods: In this descriptive cross sec...

  12. Designing Primers for H5 and H7 Subtypes of Avian Influenza Virus and Multiplex RT-PCR Amplification%禽流感病毒H5亚型及H7亚型基因的引物设计与多重RT-PCR扩增

    Institute of Scientific and Technical Information of China (English)

    张文慧; 郭华; 王伟利; 刘明; 钱爱东

    2008-01-01

    [Objective]The research aimed to design primers that are suitable for detecting H5 and H7 subtypes of avian influenza virus(AIV);[Method]DNAStar was used to analyze the homology of the sequences of H5 and H7 subtypes of AIV accessed in GenBank,and design primers(by Primer Premier 5.0) on high homologous region of these sequences,and then amplified by RT-PCR.[Result]The multiplex RT-PCR amplification,agarose gel electrophoresis and sequencing results showed that the self-designed primers are successful for detecting AIV.[Conclusion]It is feasible to rapidly diagnose AIV through this method.

  13. Imaging of Radiation Dose for Stereotactic Radiosurgery.

    Science.gov (United States)

    Guan, Timothy Y; Almond, Peter R; Park, Hwan C; Lindberg, Robert D; Shields, Christopher B

    2015-01-01

    The distributions of radiation dose for stereotactic radiosurgery, using a modified linear accelerator (Philips SL-25 and SRS-200), have been studied by using three different dosimeters: (1) ferrous-agarose-xylenol orange (FAX) gels, (2) TLD, and (3) thick-emulsion GafChromic dye film. These dosimeters were loaded into a small volume of defect in a phantom head. A regular linac stereotactic radiosurgery treatment was then given to the phantom head for each type of dosimeter. The measured radiation dose and its distributions were found to be in good agreement with those calculated by the treatment planning computer. PMID:27421869

  14. Analysis of Unconventional Approaches for the Rapid Detection of Surface Lectin Binding Ligands on Human Cell Lines

    OpenAIRE

    Welty, Lily Anne Y.; Heinrich, Eileen L.; Garcia, Karina; Banner, Lisa R.; Summers, Michael L.; Baresi, Larry; Metzenberg, Stan; Coyle-Thompson, Cathy; Oppenheimer, Steven B.

    2006-01-01

    This laboratory has developed and used a novel histochemical assay using derivatized agarose beads for over a decade to examine the surface properties of various cell types. Most recently we have used this assay to examine lectin binding ligands on two human cell types, CCL-220, a colon cancer cell line, and CRL-1459, a non-cancer colon cell line. We found that CCL-220 cells bound specific lectins better than CRL-1459, and this information was used to test for possible differential toxicity o...

  15. Human Protein Z.

    OpenAIRE

    Broze, G J; Miletich, J P

    1984-01-01

    Protein Z was purified from human plasma by a four-step procedure which included barium citrate adsorption, ammonium sulfate fractionation, DEAE-Sepharose chromatography, and blue agarose chromatography with a yield of 20%. It is a 62,000 mol wt protein with an extinction coefficient of 12.0. The concentration of Protein Z in pooled, citrated plasma is 2.2 micrograms/ml and its half-life in patients starting warfarin anticoagulation therapy is estimated to be less than 2.5 d. The NH2-terminal...

  16. Electron microscopic and DNA gel electrophoretic studies on apoptosis in HL-60 cells induced by 147Pm

    International Nuclear Information System (INIS)

    At present, the apoptosis in human leukemia HL-60 cells was studied after irradiation with 147Pm. The cumulative radiation absorption dose of 147Pm in cultural cells through different periods was estimated. The morphological changes observed under electron microscope indicated that HL-60 cells after exposure to 147Pm displayed nuclear fragmentation and nuclear margination, as well as the membrane bounded apoptotic body formation. The DNA agarose gel electrophoretic observations showed the DNA ladder pattern formation in HL-60 cells. The experimental results showed that apoptosis induced by 147Pm in HL-60 cells was dependent on the 147Pm treated time

  17. Interaction of the Chlamydia trachomatis histone H1-like protein (Hc1) with DNA and RNA causes repression of transcription and translation in vitro

    DEFF Research Database (Denmark)

    Pedersen, LB; Birkelund, Svend; Christiansen, Gunna

    1994-01-01

    The 18 kDa histone H1-like protein from Chlamydia trachomatis (Hc1) is a DNA-binding protein thought to be involved in condensation of the chlamydial chromosome during late stages in the chlamydial life cycle. Expression of Hc1 in Escherichia coli results in an overall relaxation of DNA and sever...... concentrations. These results were found to coincide with the formation of condensed Hc1-DNA and Hc1-RNA complexes as revealed by agarose gel electrophoresis and electron microscopy. The implications of these results for possible functions of Hc1 in vivo are discussed....

  18. Constructing a proton titration curve from ion-step measurements, applied to a membrane with adsorbed protein

    OpenAIRE

    Eijkel, Jan C. T.; Bosch, Coen; Olthuis, Wouter; Bergveld, Piet

    1997-01-01

    A new measuring method is described for obtaining a proton titration curve. The curve is obtained from a microporous composite membrane, consisting of polystyrene beads in an agarose matrix, with lysozyme molecules adsorbed to the bead surface. The membrane is incorporated into a sensor system by deposition on a silicon chip with a pH-sensitive ion-sensitive field effect transistor (ISFET) located in the middle of a Ag/AgCl electrode. The actual measurement is performed by creating a stepwise...

  19. DNA CLEVAGE STUDIES OF THE ETHANOLIC EXTRACTS OF THE BARK OF BAUHINIA TOMENTOSA L. AND THE WHOLE PLANT OF MUSSAENDA FRONDOSA L.

    Directory of Open Access Journals (Sweden)

    S. Gopalakrishnan

    2011-09-01

    Full Text Available DNA cleavage studies of the ethanolic extracts of the bark of Bauhinia tomentosa L. (Fam, Fabaceae and the whole plant of Mussaenda frondosa L. (Fam, Rubiaceae have been performed. The cleavage of pUC18 DNA was evaluated by agarose gel electrophoresis. Both the plant extracts show a dose dependent increase from lower concentration to higher concentration. Ethanol extract of Bauhinia tomentosa L showed more cleavage activity than that of the Mussaenda frondosa L. The results have been compared with the standard DNA cleavage agent FeSO4.

  20. In-gel DNA radiolabelling and two-dimensional pulsed field gel electrophoresis procedures suitable for fingerprinting and mapping small eukaryotic genomes

    OpenAIRE

    Brugère, Jean-François; Cornillot, Emmanuel; Méténier, Guy; Vivarès, Christian P.

    2000-01-01

    A simple method for complete genome radiolabelling is described, involving long-wave UV exposure of agarose-embedded chromosomal DNA and [α-32P]dCTP incorporation mediated by the Klenow fragment. Experiments on the budding yeast genome show that the labelling procedure can be coupled with two new two-dimensional pulsed field gel electrophoresis (2D-PFGE) protocols of genome analysis: (i) the KARD (karyotype and restriction display)-PFGE which provides a complete view of the fragments resultin...

  1. The effect of DNA concentration on mobility in pulsed field gel electrophoresis.

    OpenAIRE

    Doggett, N A; Smith, C. L.; Cantor, C R

    1992-01-01

    The effect of DNA concentration on pulsed field gel electrophoretic mobility was studied for human genomic DNA prepared in agarose inserts at 8-800 micrograms/ml and digested to completion with Not I. An eighth of each 100 microliter insert was used to produce DNA loads of 0.1 to 10 micrograms per lane. The mobility of single copy restriction fragments, as detected by hybridization, was largely concentration independent when DNA concentrations were 80 micrograms/ml or less. However, at DNA co...

  2. Replication of mitochondrial DNA occurs by strand displacement with alternative light-strand origins, not via a strand-coupled mechanism

    OpenAIRE

    Brown, Timothy A.; Cecconi, Ciro; Tkachuk, Ariana N.; Bustamante, Carlos; Clayton, David A.

    2005-01-01

    The established strand-displacement model for mammalian mitochondrial DNA (mtDNA) replication has recently been questioned in light of new data using two-dimensional (2D) agarose gel electrophoresis. It has been proposed that a synchronous, strand-coupled mode of replication occurs in tissues, thereby casting doubt on the general validity of the “orthodox,” or strand-displacement model. We have examined mtDNA replicative intermediates from mouse liver using atomic force microscopy and 2D agar...

  3. Detection of specific DNA using a microfluidic device featuring tethered poly(N-isopropylacrylamide) on a silicon substrate

    Science.gov (United States)

    Chen, Jem-Kun; Li, Jun-Yan

    2010-08-01

    In this study, we grafted thermoresponsive poly(N-isopropylacrylamide) (PNIPAAm) onto a Si substrate as the medium in a microfluidic device to detect specific DNA molecules [human genomic DNA (hgDNA528), 528 bp] at extremely low concentrations (down to 2 ng/μl). After using the polymerase chain reaction to amplify the released human gDNA signal from the tethered PNIPAAm on the substrate, the amplified human gDNA molecules were characterized through agarose gel electrophoresis. The tethered PNIPAAm in the fluid device allowed the precise detection of the human gDNA.

  4. Gel Electrophoresis of DNA - New Measurements and the Repton Model at High Fields

    International Nuclear Information System (INIS)

    New experimental data are presented on the gel electrophoresis of DNA. Experiment was made for molecules of length 173 kbp, in 1 percent agarose gel, in TAE 1 x buffer and the field intensity between 5 and 9 V/cm. The results are compared with our computer simulations, performed within the repton model of Duke and Rubinstein. The ranges of field and molecule length are determined, where the geometration effect appears. We investigate also the field dependence of the velocity and the diffusion coefficient at the border of the geometration regime. (author)

  5. Detection of Human Herpesvirus 7 DNA by Loop-Mediated Isothermal Amplification

    OpenAIRE

    Yoshikawa, Tetsushi; Ihira, Masaru; Akimoto, Shiho; Usui, Chie; Miyake, Fumi; Suga, Sadao; Enomoto, Yoshihiko; Suzuki, Ryota; Nishiyama, Yukihiro; Asano, Yoshizo

    2004-01-01

    The reliability of loop-mediated isothermal amplification (LAMP), initially developed for the detection of human herpesvirus 7 (HHV-7), was evaluated in this study. Although a LAMP product was detected in HHV-7 DNA, neither HHV-6 nor human cytomegalovirus DNA produced a product. When agarose gel electrophoresis was used for the detection of LAMP products, the sensitivity of a 30-min HHV-7 LAMP reaction reached 250 copies/tube. The use of turbidity for the detection of the LAMP products gave a...

  6. VUV irradiation studies of plasmid DNA in aqueous solution

    Science.gov (United States)

    Śmialek, M. A.; Hoffmann, S. V.; Folkard, M.; Prise, K. M.; Shuker, D. E. G.; Braithwaite, N. S.; Mason, N. J.

    2008-02-01

    Interactions of VUV light and DNA samples in aqueous solutions are reported. The damage induced by such radiation is quantified by monitoring both loss of supercoiled DNA and formation of single and double strand breaks using agarose gel electrophoresis. Irradiations were performed using synchrotron VUV photons of 130, 150, 170 and 190 nm. VUV irradiation experiments revealed enhanced damage upon irradiation with 170 nm photons as compared with irradiations with photons of 150 nm and 130 nm. Irradiations carried at 190 nm caused the least damage.

  7. DNA-directed self-assembly of gold nanoparticles into binary and ternary nanostructures

    Science.gov (United States)

    Yao, Hui; Yi, Changqing; Tzang, Chi-Hung; Zhu, Junjie; Yang, Mengsu

    2007-01-01

    The assembly and characterization of gold nanoparticle-based binary and ternary structures are reported. Two strategies were used to assemble gold nanoparticles into ordered nanoscale architectures: in strategy 1, gold nanoparticles were functionalized with single-strand DNA (ssDNA) first, and then hybridized with complementary ssDNA-labelled nanoparticles to assemble designed architectures. In strategy 2, the designed architectures were constructed through hybridization between complementary ssDNA first, then by assembling gold nanoparticles to the scaffolding through gold-sulfur bonds. Both TEM measurements and agarose gel electrophoresis confirmed that the latter strategy is more efficient in generating the designed nanostructures.

  8. A kinetic analysis of strand breaks on large DNA induced by cigarette smoke extract

    Science.gov (United States)

    Kurita, Hirofumi; Takata, Tatsuya; Yasuda, Hachiro; Takashima, Kazunori; Mizuno, Akira

    2010-06-01

    We report a kinetic analysis of strand breakages on large DNA molecules induced by cigarette smoke extract (CSE), an extract of soluble cigarette smoke components. Previously, this DNA damage was analyzed by agarose gel electrophoresis, whereas we used fluorescence to kinetically analyze damage to individual DNA molecules. CSE caused a marked change in length of DNA molecules. The rate of CSE-induced double-strand breakage on large random-coiled DNA molecules was determined using a simple theoretical model, allowing the facile estimation of the rate of double-strand breaks on large DNA molecules.

  9. Multilocus enzyme electrophoresis study of Bacillus sphaericus

    Directory of Open Access Journals (Sweden)

    Viviane Zahner

    1995-02-01

    Full Text Available Multilocus enzyme electrophoresis (MLEE has been used in the study of some Bacillus species. In this work we applied MLEE and numerical analysis in the study of the Bacillus sphaericus group. B. sphaericus can be distinguished from other entomopathogenic Bacillus by a unique allele (NP-4. Within the species, all insect pathogens were recovered in the same phenetic cluster and all of these strains have the same band position (electrophoresis migration on the agarose gel (ADH-2. The entomopathogenic group of B. sphaericus seems to be a clonal population, having two widespread frequent genotypes (zymovar 59 and zymovar 119.

  10. Resonance Energy Transfer between protein and rhamnolipid capped ZnS quantum dots: Application in in-gel staining of proteins

    Science.gov (United States)

    Janakiraman, Narayanan; Mohan, Abhilash; Kannan, Ashwin; Pennathur, Gautam

    The interaction of proteins with quantum dots is an interesting field of research. These interactions occur at the nanoscale. We have probed the interaction of Bovine Serum Albumin (BSA) and Candida rugosa lipase (CRL) with rhamnolipid capped ZnS (RhlZnSQDs) using absorption and fluorescence spectroscopy. Optical studies on mixtures of RhlZnSQDs and proteins resulted in Förster's Resonance Energy Transfer (FRET) from proteins to QDs. This phenomenon has been exploited to detect proteins in agarose gel electrophoresis. The activity of the CRL was unaffected on the addition of QDs as revealed by zymography.

  11. Comparative Study Of Native And Fructose Glycated Human Placental DNA

    OpenAIRE

    Mustafa, Imran; Garg, Nita; Raghushaker, C. R.

    2014-01-01

    Objective: The aim of our study was to glycate human placental DNA with Fructose and conduct a comparative study of properties of native and glycated DNA on the basis of UV spectrometry, fluorescence, and agarose gel electrophoresis. Methodology: Human placental DNA (10µg/ml) was incubated with 25mM fructose for 5, 10 and 15 days in phosphate buffer .Absorption profile and fluorescence emission spectra of native and glycated DNA samples were recorded. Native and glycated DNA was run on 0.8% a...

  12. Surface area of lipid membranes regulates the DNA-binding capacity of cationic liposomes

    Science.gov (United States)

    Marchini, Cristina; Montani, Maura; Amici, Augusto; Pozzi, Daniela; Caminiti, Ruggero; Caracciolo, Giulio

    2009-01-01

    We have applied electrophoresis on agarose gels to investigate the DNA-binding capacity of cationic liposomes made of cationic DC-cholesterol and neutral dioleoylphosphatidylethanolamine as a function of membrane charge density and cationic lipid/DNA charge ratio. While each cationic liposome formulation exhibits a distinctive DNA-protection ability, here we show that such a capacity is universally regulated by surface area of lipid membranes available for binding in an aspecific manner. The relevance of DNA protection for gene transfection is also discussed.

  13. Plasminogen Activator Inhibitor-1 and Susceptibility to Lung Cancer: A Population Genetics Perspective

    OpenAIRE

    Bayramoglu, Aysegul; Gunes, Hasan Veysi; Metintas, Muzaffer; Degirmenci, Irfan; Guler, Halil Ibrahim; Ustuner, Cengiz; Musmul, Ahmet

    2014-01-01

    Aim: The aim of this study was to investigate the polymorphism frequency of plasminogen activator inhibitor-1 (PAI-1) (rs1799889) 4G/5G in patients with lung cancer. Methods: In this study, 286 genomic DNAs (154 lung cancer patients+132 subjects without lung cancer) were analyzed. Polymorphisms were determined by using the polymerase chain reaction (PCR) method, with 4G and 5G allele-specific primers. PCR products were assessed by a charge-coupled device camera and exposed to 2% agarose gel e...

  14. Neutron scattering studies of the dynamics of biopolymer-water systems using pulsed-source spectrometers

    Energy Technology Data Exchange (ETDEWEB)

    Middendorf, H.D. [Univ. of Oxford (United Kingdom); Miller, A. [Stirling Univ., Stirling (United Kingdom)

    1994-12-31

    Energy-resolving neutron scattering techniques provide spatiotemporal data suitable for testing and refining analytical models or computer simulations of a variety of dynamical processes in biomolecular systems. This paper reviews experimental work on hydrated biopolymers at ISIS, the UK Pulsed Neutron Facility. Following an outline of basic concepts and a summary of the new instrumental capabilities, the progress made is illustrated by results from recent experiments in two areas: quasi- elastic scattering from highly hydrated polysaccharide gels (agarose and hyaluronate), and inelastic scattering from vibrational modes of slightly hydrated collagen fibers.

  15. Evaluation of a Novel Heminested PCR Assay Based on the Phosphoglucosamine Mutase Gene for Detection of Helicobacter pylori in Saliva and Dental Plaque

    OpenAIRE

    Goosen, C.; Theron, J.; Ntsala, M.; Maree, F F; Olckers, A; Botha, S. J.; Lastovica, A. J.; van der Merwe, S. W.

    2002-01-01

    A novel heminested PCR protocol was developed for the specific detection of Helicobacter pylori at low copy numbers. A set of primers specific for the phosphoglucosamine mutase gene (glmM) of H. pylori produced a 765-bp fragment that was used as template for the heminested primer pair delineating a 496-bp fragment. By using agarose gel electrophoresis for detection of the heminested PCR-amplified products, amplification of H. pylori genomic DNA was achieved at concentrations as low as 0.1 pg,...

  16. Synthesis of plus- and minus-strand RNA in rotavirus-infected cells.

    OpenAIRE

    Stacy-Phipps, S; Patton, J T

    1987-01-01

    The genomes of the rotaviruses consist of 11 segments of double-stranded RNA. During RNA replication, the viral plus-strand RNA serves as the template for minus-strand RNA synthesis. To characterize the kinetics of RNA replication, the synthesis and steady-state levels of viral plus- and minus-strand RNA and double-stranded RNA in simian rotavirus SA11-infected MA104 cells were analyzed by electrophoresis on 1.75% agarose gels containing 6 M urea (pH 3.0). Synthesis of viral plus-strand and m...

  17. Effect of gamma radiation on the leakage of substances from lymphocytes. In vitro study using immuno-precipitation techniques in gel medium

    International Nuclear Information System (INIS)

    In vitro effect of a 5000 R irradiation on rat blood lymphocytes was studied during several days in surviving cell suspensions, at various incubation temperature. Immunochemical analysis in agarose gel, using the simple diffusion technique, exhibited the leakage of two groups of antigenic compounds, from lymphocytes. The first group compounds seemed to be periodical renewal products of surface cell membrane elements, common to lymphocytes and erythrocytes; irradiation increased their release. A correlation was established between the second group compounds and cell metabolism and these compounds seemed to be enzymes of cytolitic origin

  18. Spheroid control of malignant glioma cell lines after fractionated irradiation

    International Nuclear Information System (INIS)

    Spheroid control doses (SCD50) were determined for ten human glioma lines after fractionated irradiation under oxic conditions. In addition, SF2 values and colony forming efficiencies (CFE) were measured in a soft agarose clonogenic assay. A significant relationship existed between the SCD50 values and the SF2CFE data pairs (p=0.01) but the SCD50 values were higher than expected from the SF2 and CFE values. This comparison shows the influence of environmental factors (different in both model systems) on reproductive tumour cell death after irradiation. (author). figs., tab

  19. Water Extract of Samultang Reduces Apoptotic Cell Death by H2O2-Induced Oxidative Injury in SK-N-MC Cells

    OpenAIRE

    Lee, Gyoung Wan; Kim, Min Sun

    2009-01-01

    The purpose of this study was to evaluate the effects of the water extract of Samultang (SMT), a Chinese herb, on apoptotic cell death by H2O2-induced oxidative stress in SK-N-MC cells. A nuclear fragmentation was observed via fluorescence imaging 12 h after exposure to 30 µM H2O2 and DNA laddering was detected via agarose electrophoresis gel. In addition, increases in sub-G1 phase and cleavage of the PARP protein were observed. However, treatment with SMT for 2 h prior to H2O2 exposure signi...

  20. Enzymatic Ligation Creates Discrete Multi-Nanoparticle Building Blocks for Self-Assembly

    Energy Technology Data Exchange (ETDEWEB)

    Claridge, Shelley A.; Mastroianni, Alexander J.; Au, Yeung B.; Liang, Huiyang W.; Micheel, Christine M.; Frechet, Jean M.J.; Alivisatos, A. Paul

    2008-05-27

    Enzymatic ligation of discrete nanoparticle?DNA conjugates creates nanoparticle dimer and trimer structures in which the nanoparticles are linked by single-stranded DNA, rather than double-stranded DNA as in previous experiments. Ligation is verified by agarose gel and small-angle X-ray scattering. This capability is utilized in two ways: first to create a new class of multiparticle building blocks for nanoscale self-assembly; second to develop a system which can amplify a population of discrete nanoparticle assemblies.