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31.
Rapid, continuous purification of proteins in a microfluidic device using genetically-engineered partition tags 总被引:2,自引:0,他引:2
High-throughput screening assays of native and recombinant proteins are increasingly crucial in life science research, including fields such as drug screening and enzyme engineering. These assays are typically highly parallel, and require minute amounts of purified protein per assay. To address this need, we have developed a rapid, automated microscale process for isolating specific proteins from sub-microlitre volumes of E. Coli cell lysate. Recombinant proteins are genetically tagged to drive partitioning into the PEG-rich phase of a flowing aqueous two-phase system, which removes approximately 85% of contaminating proteins, as well as unwanted nucleic acids and cell debris, on a simple microfluidic device. Inclusion of the genetic tag roughly triples recovery of the autofluorescent protein AcGFP1, and also significantly improves recovery of the enzyme glutathione S-transferase (GST), from nearly zero recovery for the wild-type enzyme, up to 40% with genetic tagging. The extraction process operates continuously, with only a single step from cell lysate to purified protein, and does not require expensive affinity reagents or troublesome chromatographic steps. The two-phase system is mild and does not disrupt protein function, as evidenced by recovery of active enzymes and functional fluorescent protein from our microfluidic process. The microfluidic aqueous two-phase extraction forms the core component of an integrated lab-on-a-chip device comprising cell culture, lysis, purification and analysis on a single device. 相似文献
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Zendejas FJ Meagher RJ Stachowiak JC Hayden CC Sasaki DY 《Chemical communications (Cambridge, England)》2011,47(26):7320-7322
Directing the orientation of molecular assemblies is a key step toward creating complex hierarchical structures that yield higher order functional materials. Here, we demonstrate the directed orientation of functionalized lipid domains and protein-membrane assemblies, using an electric field. 相似文献
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Mark T McAuley Rose Anne Kenny Thomas BL Kirkwood Darren J Wilkinson Janette JL Jones Veronica M Miller 《BMC neuroscience》2009,10(1):26
Background
The hippocampus is essential for declarative memory synthesis and is a core pathological substrate for Alzheimer's disease (AD), the most common aging-related dementing disease. Acute increases in plasma cortisol are associated with transient hippocampal inhibition and retrograde amnesia, while chronic cortisol elevation is associated with hippocampal atrophy. Thus, cortisol levels could be monitored and managed in older people, to decrease their risk of AD type hippocampal dysfunction. We generated an in silicomodel of the chronic effects of elevated plasma cortisol on hippocampal activity and atrophy, using the systems biology mark-up language (SBML). We further challenged the model with biologically based interventions to ascertain if cortisol associated hippocampal dysfunction could be abrogated. 相似文献38.
The kinetics of the reaction of O + CH3OCH3 were investigated using fast-flow apparatus equipped with ESR and mass-spectrometric detection. The concentration of O(3P) atoms to CH3OCH3 was varied over an unusually large range. The rate constant for reaction was found to be k = (5.0 ± 1.0) × 1012 exp [(?2850 ± 200/RT)] cm3 mole?1 sec?1. The reaction O + CH3OH was studied using ESR detection. Based on an assumed stoichiometry of two oxygen atoms consumed per molecule of CH3OH which reacts, we obtain a value of k = (1.70 ± 0.66) × 1012 exp [(?2,280 ± 200/RT)] cm3 mole?1 sec?1 for the reaction The results obtained in this study are compared with the results from other workers on these reactions. The observation of essentially equal activation energies in these two reactions is indicative of approximately equal C? H bond strengths in CH3OCH3 and CH3OH. This is in agreement with recent measurements of these bond energies. 相似文献
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Relevant equivalent circuit parameters and values of material constants of a piezoelectric resonator can be determined from measurements of its electrical input impedance as a function of frequency. The complex electrical impedance curves and the associated critical frequencies are the basis of this characterization by the piezoelectric resonance method. In this paper, the previously introduced concept of normalized electrical impedance of the lossy resonator, extended to include piezoelectric losses, is applied to the analysis of the effects of different types of intrinsic losses on peak values, bandwidths and characteristic frequencies. The resulting impedance patterns depend solely on the electromechanical coupling coefficient and the loss tangents, providing a useful tool for the analysis of low-Q resonators. The normalized impedance is experimentally evaluated from the basic data provided by an HP 4194A impedance analyser by means of specifically developed ASP programs. 相似文献
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Covering arrays have applications in software, network and circuit testing. In this article, we consider a generalization of covering arrays that allows mixed alphabet sizes as well as a graph structure that specifies the pairwise interactions that need to be tested. Let k and n be positive integers, and let G be a graph with k vertices v1,v2,…, vk with respective vertex weights g1 ≤ g2 ≤ … ≤ gk. A mixed covering array on G, denoted by , is an n × k array such that column i corresponds to vi, cells in column i are filled with elements from ?gi and every pair of columns i,j corresponding to an edge vi,vj in G has every possible pair from ?gi × ?gj appearing in some row. The number of rows in such array is called its size. Given a weighted graph G, a mixed covering array on G with minimum size is called optimal. In this article, we give upper and lower bounds on the size of mixed covering arrays on graphs based on graph homomorphisms. We provide constructions for covering arrays on graphs based on basic graph operations. In particular, we construct optimal mixed covering arrays on trees, cycles and bipartite graphs; the constructed optimal objects have the additional property of being nearly point balanced. © 2007 Wiley Periodicals, Inc. J Combin Designs 15: 393–404, 2007 相似文献