971 resultados para Electrophoresis, Polyacrylamide Gel


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Ternary iron(III) complexes (FeL(B)] (1-3) of a trianionic tetradentate phenolate-based ligand (L) and henanthroline base (B), namely, 1,10-phenanthroline (phen, 1), dipyridoquinoxaline (dpq, 2), and dipyridophenazine (dppz, 3), have been prepared and structurally characterized and their DNA binding, cleavage, and photocytotoxic properties studied. The complexes with a FeN3O3 core show the Fe(III)/Fe(II) redox couple near -0.6 V in DMF, a magnetic moment value of similar to 5.9 mu(B), and a binding propensity to both calf thymus DNA and bovine serum albumin (BSA) protein. They exhibit red-light-induced DNA cleavage activity following a metal-assisted photoredox pathway forming HO center dot radicals but do not show any photocleavage of BSA in UV-A light. Complex 3 displays photocytotoxicity in the human cervical cancer cell line (HeLa) and human keratinocyte cell line (HaCaT) with respective IC50 values of 3.59 mu M and 6.07 mu M in visible light and 251 nM and 751 nM in UV-A light of 365 nm. No significant cytotoxicity is observed in the dark. The photoexposed HeLa cells, treated prior with complex 3, have shown marked changes in nuclear morphology as demonstrated by Hoechst 33258 nuclear stain. Generation of reactive oxygen species has been evidenced from the fluorescence enhancement of dichlorofluorescein upon treatment with 3 followed by photoexposure. Nuclear chromatin cleavage has been observed in acridine orange/ethidium bromide dual staining of treated HeLa cells and from alkaline single-cell gel electrophoresis. Caspase 3/7 activity in HeLa cells has been found to be upregulated by only 4 fold after photoirradiation, signifying the fact that cell death through a caspase 3/7 dependent pathway may not be solely operative.

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TiO2 films are extensively used in various applications including optical multi-layers, sensors, photo catalysis, environmental purification, and solar cells etc. These are prepared by both vacuum and non-vacuum methods. In this paper, we present the results on TiO2 thin films prepared by a sol-gel spin coating process in non-aqueous solvent. Titanium isopropoxide is used as TiO2 precursor. The films were annealed at different temperatures up to 3000 C for 5 hours in air. The influence of the various deposition parameters like spinning speed, spinning time and annealing temperature on the thickness of the TiO2 films has been studied. The variation of film thickness with time in ambient atmosphere was also studied. The optical, structural and morphological characteristics were investigated by optical transmittance-reflectance measurements, X-ray diffraction (XRD) and scanning electron microscopy (SEM) respectively. The refractive index and extinction coefficient of the films were determined by envelope technique and spectroscopic ellipsometry. TiO2 films exhibited high transparency (92%) in the visible region with a refractive index of 2.04 at 650 nm. The extinction coefficient was found to be negligibly small. The X-ray diffraction analysis showed that the TiO2 film deposited on glass substrate changes from amorphous to crystalline (anatase) phase with annealing temperature above 2500 C. SEM results show that the deposited films are uniform and crack free.

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An inexpensive and effective simple method for the preparation of nano-crystalline titanium oxide (anatase) thin films at room temperature on different transparent substrates is presented. This method is based on the use of peroxo-titanium complex, i.e. titanium isopropoxide as a single initiating organic precursor. Post-annealing treatment is necessary to convert the deposited amorphous film into titanium oxide (TiO2) crystalline (anatase) phase. These films have been characterized for X-ray diffraction (XRD) studies, atomic force microscopic (AFM) studies and optical measurements. The optical constants such as refractive index and extinction coefficient have been estimated by using envelope technique. Also, the energy gap values have been estimated using Tauc's formula for on glass and quartz substrates are found to be 3.35 eV and 3.39 eV, respectively. (C) 2008 Elsevier B.V. All rights reserved.

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Miniaturization of analytical instrumentation is attracting growing interest in response to the explosive demand for rapid, yet sensitive analytical methods and low-cost, highly automated instruments for pharmaceutical and bioanalyses and environmental monitoring. Microfabrication technology in particular, has enabled fabrication of low-cost microdevices with a high degree of integrated functions, such as sample preparation, chemical reaction, separation, and detection, on a single microchip. These miniaturized total chemical analysis systems (microTAS or lab-on-a-chip) can also be arrayed for parallel analyses in order to accelerate the sample throughput. Other motivations include reduced sample consumption and waste production as well as increased speed of analysis. One of the most promising hyphenated techniques in analytical chemistry is the combination of a microfluidic separation chip and mass spectrometer (MS). In this work, the emerging polymer microfabrication techniques, ultraviolet lithography in particular, were exploited to develop a capillary electrophoresis (CE) separation chip which incorporates a monolithically integrated electrospray ionization (ESI) emitter for efficient coupling with MS. An epoxy photoresist SU-8 was adopted as structural material and characterized with respect to its physicochemical properties relevant to chip-based CE and ESI/MS, namely surface charge, surface interactions, heat transfer, and solvent compatibility. As a result, SU-8 was found to be a favorable material to substitute for the more commonly used glass and silicon in microfluidic applications. In addition, an infrared (IR) thermography was introduced as direct, non-intrusive method to examine the heat transfer and thermal gradients during microchip-CE. The IR data was validated through numerical modeling. The analytical performance of SU-8-based microchips was established for qualitative and quantitative CE-ESI/MS analysis of small drug compounds, peptides, and proteins. The CE separation efficiency was found to be similar to that of commercial glass microchips and conventional CE systems. Typical analysis times were only 30-90 s per sample indicating feasibility for high-throughput analysis. Moreover, a mass detection limit at the low-attomole level, as low as 10E+5 molecules, was achieved utilizing MS detection. The SU-8 microchips developed in this work could also be mass produced at low cost and with nearly identical performance from chip to chip. Until this work, the attempts to combine CE separation with ESI in a chip-based system, amenable to batch fabrication and capable of high, reproducible analytical performance, have not been successful. Thus, the CE-ESI chip developed in this work is a substantial step toward lab-on-a-chip technology.

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Purpose A modification of the existing PVA-​FX hydrogel has been made to investigate the use of a functionalised polymer in a Fricke gel dosimetry system to decrease Fe3+ diffusion. Methods The chelating agent, xylenol orange, was chem. bonded to the gelling agent, polyvinyl alc. (PVA) to create xylenol orange functionalised PVA (XO-​PVA)​. A gel was created from the XO-​PVA (20​% w​/v) with ferrous sulfate (0.4 mM) and sulfuric acid (50 mM)​. Results This resulted in an optical d. dose sensitivity of 0.014 Gy-​1, an auto-​oxidn. rate of 0.0005 h-​1, and a diffusion rate of 0.129 mm2 h-​1; an 8​% redn. compared to the original PVA-​FX gel, which in practical terms adds approx. 1 h to the time span between irradn. and accurate read-​out. Conclusions Because this initial method of chem. bonding xylenol orange to polyvinyl alc. has inherently low conversion, the improvement on existing gel systems is minimal when compared to the drawbacks. More efficient methods of functionalising polyvinyl alc. with xylenol orange must be developed for this system to gain clin. relevance.

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The aim of this study was to compare the use of indirect haemagglutination (IHA) and gel diffusion (GD) tests for serotyping Haemophilus parasuis by the Kielstein-Rapp-Gabrielson scheme. All 15 serovar reference strains, 72 Australian field isolates, nine Chinese field isolates, and seven isolates from seven experimentally infected pigs were evaluated with both tests. With the IHA test, 14 of the 15 reference strains were correctly serotyped – with serovar 10 failing to give a titre with serovar 10 antiserum. In the GD test, 13 reference strains were correctly serotyped – with antigen from serovars 7 and 8 failing to react with any antiserum. The IHA methodology serotyped a total of 45 of 81 field isolates while the GD methodology serotyped a total of 48 isolates. For 29 isolates, the GD and IHA methods gave discordant results. It was concluded that the IHA is a good additional test for the serotyping of H. parasuis by the KRG scheme if the GD methodology fails to provide a result or shows unusual cross-reactions.

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Gossypol, a polyphenolic compound isolated from cotton plant was found to degrade pBR322 DNA Image in a reaction which required the presence of a metal ion, a reducing agent (2-mercaptoethanol) and oxygen as revealed after agarose gel electrophoresis. Fe3+ and Co2+ showed maximum degradation whereas addition of Ca2+ and Mg2+ prevented the gossypol mediated DNA damage. Gossypol caused degradation of rat liver DNA incubated Image even in the absence of added metal ions and 2-mercaptoethanol. Incubation of intact rat liver nuclei with gossypol reveled DNA degradation and nuclei isolated from rats treated with gossypol Image showed higher succestibility to DNA fragmentation when incubated with gossypol Image than control nuclei. EcoRl and AIuI digestion of DNA isolated from gossypol treated rats gave clear cut evidence for DNA degradation. These observations indicate that gossypol is genotoxic and considereable care has to be exercised in its use. SDS, sodium dodecayl sulphate; TE buffer, Tris-HCL-EDTA buffer.

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Protein fractions that bind retinol were isolated from the cytosol, nucleosol and chromatin of the oviduct magnum of laying hens. The proteins isolated from the three sources showed similar elution profiles on chromatography through Sephadex G-75 and G-50 columns, and comparable mobility during electrophoresis on sodium dodecyl sulphate/polyacrylamide gels. Their molecular weights were calculated to be around 14500. When oviducts from vitamin A-depleted and vitamin A-repleted immature chicks given oestrogen injections for 6 consecutive days were incubated with [3H]retinyl acetate, uptake of the radioactivity in the nuclei of the vitamin A-depleted tissue was severalfold higher than that in the nuclei from the vitamin A-repleted tissue.

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Immunoglobulin Y is different from most of the other immunoglobulins because it does not bind protein A or protein G. Thiophilic gel chromatography has been successfully used to purify IgY from chicken egg yolk, but the technology has not previously been used to purify IgY from serum. In this research note, we describe the optimization of T-gel chromatography for purification of IgY from serum. Data are provided on the recovery and purity of IgY obtained using potassium sulfate buffers of different concentrations. Decreasing the strength of potassium sulfate buffer from 0.5 to 0.3 M did not alter the amount of IgY recovered but increased the purity. Using 0.3 M potassium sulphate, we recovered approximately 63.7% of the serum Ig as almost pure IgY.

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Aims: To identify dominant bacteria in grain (barley)-fed cattle for isolation and future use to increase the efficiency of starch utilization in these cattle. Methods and Results: Total DNA was extracted from samples of the rumen contents from eight steers fed a barley diet for 9 and 14 days. Bacterial profiles were obtained using denaturing gradient gel electrophoresis (DGGE) of the PCR-amplified V2/V3 region of the 16S rRNA genes from total bacterial DNA. Apparently dominant bands were excised and cloned, and the clone insert sequence was determined. One of the most common and dominant bacteria present was identified as Ruminococcus bromii. This species was subsequently isolated using traditional culture-based techniques and its dominance in the grain-fed cattle was confirmed using a real-time Taq nuclease assay (TNA) designed for this purpose. In some animals, the population of R. bromii reached densities above 1010R. bromii cell equivalents per ml or approximately 10% of the total bacterial population. Conclusions: Ruminococcus bromii is a dominant bacterial population in the rumen of cattle fed a barley-based diet. Significance and Impact of the Study: Ruminococcus bromii YE282 may be useful as a probiotic inoculant to increase the efficiency of starch utilization in barley-fed cattle. The combination of DGGE and real-time TNA has been an effective process for identifying and targeting for isolation, dominant bacteria in a complex ecosystem.

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Background: With the advances in DNA sequencer-based technologies, it has become possible to automate several steps of the genotyping process leading to increased throughput. To efficiently handle the large amounts of genotypic data generated and help with quality control, there is a strong need for a software system that can help with the tracking of samples and capture and management of data at different steps of the process. Such systems, while serving to manage the workflow precisely, also encourage good laboratory practice by standardizing protocols, recording and annotating data from every step of the workflow Results: A laboratory information management system (LIMS) has been designed and implemented at the International Crops Research Institute for the Semi-Arid Tropics (ICRISAT) that meets the requirements of a moderately high throughput molecular genotyping facility. The application is designed as modules and is simple to learn and use. The application leads the user through each step of the process from starting an experiment to the storing of output data from the genotype detection step with auto-binning of alleles; thus ensuring that every DNA sample is handled in an identical manner and all the necessary data are captured. The application keeps track of DNA samples and generated data. Data entry into the system is through the use of forms for file uploads. The LIMS provides functions to trace back to the electrophoresis gel files or sample source for any genotypic data and for repeating experiments. The LIMS is being presently used for the capture of high throughput SSR (simple-sequence repeat) genotyping data from the legume (chickpea, groundnut and pigeonpea) and cereal (sorghum and millets) crops of importance in the semi-arid tropics. Conclusions: A laboratory information management system is available that has been found useful in the management of microsatellite genotype data in a moderately high throughput genotyping laboratory. The application with source code is freely available for academic users and can be downloaded from http://www.icrisat.org/bt-software-d-lims.htm

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RFLP markers are currently the most appropriate marker system for the identification of uncharacterised polymorphism at the interspecific and intergeneric level. Given the benefits of a PCR-based marker system and the availability of sequence information for many Solanaceous cDNA clones, it is now possible to target conserved fragments, for primer development, that flank sequences possessing interspecific polymorphism. The potential outcome is the development of a suite of markers that amplify widely in Solanaceae. Temperature gradient gel electrophoresis (TGGE) is a relatively inexpensive gel-based system that is suitable for the detection of most single-base changes. TGGE can be used to screen for both known and unknown polymorphisms, and has been assessed here, for the development of PCR-based markers that are useful for the detection of interspecific variation within Solanaceae. Fifteen markers are presented where differences between Lycopersicon esculentum and L. pennellii have been detected by TGGE. The markers were assessed on a wider selection of plant species and found to be potentially useful for the identification of interspecific and intergeneric polymorphism in Solanaceous plants.

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Methane emissions from ruminant livestock represent a loss of carbon during feed conversion, which has implications for both animal productivity and the environment because this gas is considered to be one of the more potent forms of greenhouses gases contributing to global warming. Many strategies to reduce emissions are targeting the methanogens that inhabit the rumen, but such an approach can only be successful if it targets all the major groups of ruminant methanogens. Therefore, a thorough knowledge of the diversity of these microbes in different breeds of cattle and sheep, as well as in response to different diets, is required. A study was undertaken using the molecular techniques denaturing gradient gel electrophoresis, DNA cloning and DNA sequence analysis to define the extent of diversity among methanogens in ruminants, particularly Bos indicus cross cattle, on differing forages in Queensland. It was found that the diversity of methanogens in forage-fed cattle in Queensland was greater than in grain-fed cattle but there was little variability in methanogen community composition between cattle fed different forages. The species that dominate the rumen microbial communities of B. indicus cross cattle are from the genus Methanobrevibacter, although rumen-fluid inoculated digestors fed Leucaena leucocephala leaf were populated with Methanosphaera-like strains, with the Methanobrevibacter-like strains displaced. If ruminant methane emissions are to be reduced, then antimethanogen bioactives that target both broad groups of ruminant methanogens are most likely to be needed, and as a part of an integrated suite of approaches that redirect rumen fermentation towards other more useful end products.