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Hui Lu Edgar Arriaga Yong Chen Da Daniel Figeys Norman J. Dovichi 《Journal of chromatography. A》1994,680(2):503-510
In DNA sequencing, single-stranded DNA fragments are separated by gel electrophoresis. This separation is based on a sieving mechanism where DNA fragments are retarded as they pass through pores in the gel. In this paper, we present the mobility of DNA sequencing fragments as a function of temperature; mobility is determined in 4% T LongRanger gels at an electric field of 300 V/cm. The temperature dependence is compared with the predictions of the biased reptation model. The model predicts that the fragment length for the onset of biased reptation with stretching increases with the square of temperature; the data show that the onset of biased reptation with stretching decreases with temperature. Biased reptation fails to model accurately the temperature dependence of mobility. We analyzed the data and extracted the activation energy for passage of sequencing fragments through the gel. For fragments containing less than ca. 200 bases, the activation energy increases linearly with the number of bases at a rate of 25 J/mol per base; for longer fragments, the activation energy increases at a rate of 6.5 J/mol per base. This transition in the activation energy presumably reflects a change in conformation of the DNA fragments; small fragments exist in a random coil configuration and larger fragments migrate in an elongated configuration. 相似文献
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Thane H. Taylor Nicholas W. Frost Michael T. Bowser Edgar A. Arriaga 《Analytical and bioanalytical chemistry》2014,406(6):1683-1691
Mitochondria are responsible for maintaining a variety of cellular functions. One such function is the interaction and subsequent import of proteins into these organelles via the translocase of outer membrane (TOM) complex. Antibodies have been used to analyze the presence and function of proteins comprising this complex, but have not been used to investigate variations in the abundance of TOM complex in mitochondria. Here, we report on the feasibility of using capillary cytometry with laser-induced fluorescence to detect mitochondria labeled with antibodies targeting the TOM complex and to estimate the number of antibodies that bind to these organelles. Mitochondria were fluorescently labeled with DsRed2, while antibodies targeting the TOM22 protein, one of nine proteins comprising the TOM complex, were conjugated to the Atto-488 fluorophore. At typical labeling conditions, 94 % of DsRed2 mitochondria were also immunofluorescently labeled with Atto-488 Anti-TOM22 antibodies. The calculated median number of Atto-488 Anti-TOM22 antibodies bound to the surface of mitochondria was ~2,000 per mitochondrion. The combination of fluorescent immunolabeling and capillary cytometry could be further developed to include multicolor labeling experiments, which enable monitoring several molecular targets at the same time in the same or different organelle types. Figure
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We determined the feasibility of using capillary electrophoresis with postcolumn laser-induced fluorescence (CE-LIF) detection to characterize electrophoretic properties of isolated cell nuclei and impurities present in nuclear fractions. These fractions were isolated from NS-1 mouse hybridoma cells, stained with hexidium iodide, a DNA intercalating dye, and analyzed by CE-LIF detection. The corresponding electropherograms display two features: (i) broad peaks (6-90 s wide) caused by the cell culturing media and by free-DNA intercalated with hexidium iodide, and (ii) a large number of narrow peaks (180 ms wide), resulting from DNA associated with individual intact or disrupted nuclei. We confirmed that the narrow peaks were not caused by contaminating mitochondria. The overall electrophoretic mobility range of disrupted nuclei is 0 to -5 x 10(-4)cm(2)/Vs, while intact nuclei seem to have mobilities in the -1.5 to -3.5 x 10(-4)cm(2)/Vs range. Furthermore, the highly sensitive CE-LIF method reveals a high abundance of disrupted nuclei that cannot be directly observed by confocal microscopy. 相似文献
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J. Ledesma-Garcia I. L. Escalante-Garcia Thomas W. Chapman L. G. Arriaga V. Baglio V. Antonucci A. S. Aricò R. Ornelas Luis A. Godinez 《Journal of Solid State Electrochemistry》2010,14(5):835-840
Dendrimer-encapsulated Pt nanoparticles (G4OHPt) were prepared by chemical reduction at room temperature. The G4OHPt, with
average diameters of ca. 2.7 nm, were characterized by X-ray diffraction, scanning electron microscopy, and thermogravimetric
analysis. Electrocatalytic behavior for oxygen reduction reaction was investigated using a rotating disk electrode configuration
in an acidic medium, with and without the presence of methanol (0.01, 0.1, and 1 M). Kinetic studies showed that electrodes
based on Pt nanoparticles encapsulated inside the dendrimer display a higher selectivity for ORR in the presence of methanol
than electrodes based on commercial Pt black catalysts. Also, the dendritic polymer confers a protective effect on the Pt
in the presence of methanol, which allows its use as a cathode in a direct methanol fuel cell operating at different temperatures.
Good performance was obtained at 90 °C and 2 bar of pressure with a low platinum loading on the electrode surface. 相似文献
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Bobby G. Poe III Ciarán F. Duffy Michael A. Greminger Bradley J. Nelson Edgar A. Arriaga 《Analytical and bioanalytical chemistry》2010,397(8):3397-3407
Mitochondrial DNA (mtDNA) mutations have been associated with disease and aging. Since each cell has thousands of mtDNA copies,
clustered into nucleoids of five to ten mtDNA molecules each, determining the effects of a given mtDNA mutation and their
connection with disease phenotype is not straightforward. It has been postulated that heteroplasmy (coexistence of mutated
and wild-type DNA) follows simple probability rules dictated by the random distribution of mtDNA molecules at the nucleoid
level. This model has been used to explain how mutation levels correlate with the onset of disease phenotype and loss of cellular
function. Nonetheless, experimental evidence of heteroplasmy at the nucleoid level is scarce. Here, we report a new method
to determine heteroplasmy of individual mitochondrial particles containing one or more nucleoids. The method uses capillary
cytometry with laser-induced fluorescence detection to detect individual mitochondrial particles stained with PicoGreen, which
makes it possible to quantify the mtDNA copy number of each particle. After detection, one or more particles are collected
into polymerase chain reaction (PCR) wells and then subjected to real-time multiplexed PCR amplification. This PCR strategy
is suitable to obtain the relative abundance of mutated and wild-type mtDNA. The results obtained here indicate that individual
mitochondrial particles and nucleoids contained within these particles are not heteroplasmic. The results presented here suggest
that current models of mtDNA segregation and distribution (i.e., heteroplasmic nucleoids) need further consideration. 相似文献
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Theresa Hague Andrea Petroczi Paul LR Andrews James Barker Declan P Naughton 《Chemistry Central journal》2010,4(1):2
This is a correction to the following paper: Hague T, Petroczi A, Andrews PR, Barker J, Naughton DP: Determination of metal
ion content of beverages and estimation of target hazard quotients: a comparative study. Chem Central J 2008, 2:13. 相似文献
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The analysis of mitochondria by capillary electrophoresis usually takes longer than 20 min per replicate which may compromise the quality of the mitochondria due to degradation. In addition, low sample consumption may be beneficial in the analysis of rare or difficult samples. In this report, we demonstrate the ability to analyze individual mitochondrial events in picoliter-volume samples (approximately 80 pL) taken from a bovine liver preparation using microchip capillary electrophoresis with laser-induced fluorescence detection (micro-chip CE-LIF). Using a commercial "double-T" glass microchip, the sample was electrokinetically loaded in the "double-T" intersection and then subjected to electrophoretic separation along the main separation channel. In order to decrease interactions of mitochondria with channel walls during the analysis, poly(vinyl alcohol) was used as a dynamic coating. This procedure eliminates the need for complicated covalent surface modifications within the channels that were previously used in capillary electrophoresis methods. For analysis, mitochondria, isolated from bovine liver tissue, were selectively labelled using 10-nonyl acridine orange (NAO). The results consist of electropherograms where each mitochondrial event is a narrow spike (240 +/- 44 ms). While the spike intensity is representative of its NAO content, its migration time is used to calculate and describe its electrophoretic mobility, which is a property still largely unexplored for intracellular organelles. The five-fold decrease in separation time (4 min for microchip versus 20 min for capillary electrophoresis) makes microchip electrophoretic separations of organelles a faster, sensitive, low-sample volume alternative for the characterization of individual organelle properties and for investigations of subcellular heterogeneity. 相似文献