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41.
Andrea LJ Marschall Congcong Zhang André Frenzel Thomas Schirrmann Michael Hust Franck Perez Stefan Dübel 《MABS-AUSTIN》2014,6(4):943-956
The use of antibodies to target their antigens in living cells is a powerful analytical tool for cell biology research. Not only can molecules be localized and visualized in living cells, but interference with cellular processes by antibodies may allow functional analysis down to the level of individual post-translational modifications and splice variants, which is not possible with genetic or RNA-based methods. To utilize the vast resource of available antibodies, an efficient system to deliver them into the cytosol from the outside is needed. Numerous strategies have been proposed, but the most robust and widely applicable procedure still remains to be identified, since a quantitative ranking of the efficiencies has not yet been done. To achieve this, we developed a novel efficiency evaluation method for antibody delivery based on a fusion protein consisting of a human IgG1 Fc and the recombination enzyme Cre (Fc-Cre). Applied to suitable GFP reporter cells, it allows the important distinction between proteins trapped in endosomes and those delivered to the cytosol. Further, it ensures viability of positive cells and is unsusceptible to fixation artifacts and misinterpretation of cellular localization in microscopy and flow cytometry. Very low cytoplasmic delivery efficiencies were found for various profection reagents and membrane penetrating peptides, leaving electroporation as the only practically useful delivery method for antibodies. This was further verified by the successful application of this method to bind antibodies to cytosolic components in living cells. 相似文献
42.
Mario PL Calus Theo HE Meuwissen Jack J Windig Egbert F Knol Chris Schrooten Addie LJ Vereijken Roel F Veerkamp 《遗传、选种与进化》2009,41(1):11
The aim of this paper was to compare the effect of haplotype definition on the precision of QTL-mapping and on the accuracy of predicted genomic breeding values. In a multiple QTL model using identity-by-descent (IBD) probabilities between haplotypes, various haplotype definitions were tested i.e. including 2, 6, 12 or 20 marker alleles and clustering base haplotypes related with an IBD probability of > 0.55, 0.75 or 0.95. Simulated data contained 1100 animals with known genotypes and phenotypes and 1000 animals with known genotypes and unknown phenotypes. Genomes comprising 3 Morgan were simulated and contained 74 polymorphic QTL and 383 polymorphic SNP markers with an average r2 value of 0.14 between adjacent markers. The total number of haplotypes decreased up to 50% when the window size was increased from two to 20 markers and decreased by at least 50% when haplotypes related with an IBD probability of > 0.55 instead of > 0.95 were clustered. An intermediate window size led to more precise QTL mapping. Window size and clustering had a limited effect on the accuracy of predicted total breeding values, ranging from 0.79 to 0.81. Our conclusion is that different optimal window sizes should be used in QTL-mapping versus genome-wide breeding value prediction. 相似文献
43.
Accumulation of glucosinolates, a class of defense-related secondary metabolites found almost exclusively in the Capparales, is induced in response to a variety of biological stresses. It is often assumed that elevated glucosinolate levels result from de novo biosynthesis, but glucosinolate transport from other parts of the plant to the site of herbivory or pathogen infection can also contribute to the defense response. Several studies with Arabidopsis and other crucifers have demonstrated that glucosinolates from vegetative tissue are transported to developing seeds. Here we discuss evidence that long-chain aliphatic glucosinolates are transported to the site of herbivory in response to Myzus persicae (green peach aphid) feeding on Arabidopsis.Key Words: glucosinolate, transport, graft, Arabidopsis, Myzus persicae, aphid 相似文献
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45.
Sheffield PJ McMullen TW Li J Ho YS Garrard SM Derewenda U Derewenda ZS 《Protein engineering》2001,14(7):513-519
The intracellular form of mammalian platelet activating factor acetylhydrolase found in brain (PAF-AH Ib) is thought to play a critical role in control in neuronal migration during cortex development. This oligomeric complex consists of a homodimer of the 45 kDa (beta) LIS1 protein, the product of the causative gene for type I lissencephaly, and, depending on the developmental stage and species, one of three possible pairs of two homologous approximately 26 kDa alpha-subunits, which harbor all of the catalytic activity. The exact composition of this complex depends on the expression patterns of the alpha(1) and alpha(2) genes, exhibiting tissue specificity and developmental control. All three possible dimers (alpha(1)/alpha(1), alpha(1)/alpha(2) and alpha(2)/alpha(2)) were identified in tissues. The alpha(1)/alpha(2) heterodimer is thought to play an important role in fetal brain. The structure of the alpha(1)/alpha(1) homodimer was solved earlier in our laboratory at 1.7 A. We report here the preparation of recombinant alpha(1)/alpha(2) heterodimers using a specially constructed bi-cistronic expression vector. The approach may be useful in studies of other systems where pure heterodimers of recombinant proteins are required. The alpha(1)/alpha(2) dimer has been crystallized and its structure was solved at 2.1 A resolution by molecular replacement. These results set the stage for a detailed characterization of the PAF-AH Ib complex. 相似文献
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48.
Dave Kendal Cindy E. Hauser Georgia E. Garrard Sacha Jellinek Katherine M. Giljohann Joslin L. Moore 《PloS one》2013,8(8)
Human perception of plant leaf and flower colour can influence species management. Colour and colour contrast may influence the detectability of invasive or rare species during surveys. Quantitative, repeatable measures of plant colour are required for comparison across studies and generalisation across species. We present a standard method for measuring plant leaf and flower colour traits using images taken with digital cameras. We demonstrate the method by quantifying the colour of and colour difference between the flowers of eleven grassland species near Falls Creek, Australia, as part of an invasive species detection experiment. The reliability of the method was tested by measuring the leaf colour of five residential garden shrub species in Ballarat, Australia using five different types of digital camera. Flowers and leaves had overlapping but distinct colour distributions. Calculated colour differences corresponded well with qualitative comparisons. Estimates of proportional cover of yellow flowers identified using colour measurements correlated well with estimates obtained by measuring and counting individual flowers. Digital SLR and mirrorless cameras were superior to phone cameras and point-and-shoot cameras for producing reliable measurements, particularly under variable lighting conditions. The analysis of digital images taken with digital cameras is a practicable method for quantifying plant flower and leaf colour in the field or lab. Quantitative, repeatable measurements allow for comparisons between species and generalisations across species and studies. This allows plant colour to be related to human perception and preferences and, ultimately, species management. 相似文献
49.
Stoichiometry of chromatin proteins 总被引:7,自引:0,他引:7
W T Garrard W R Pearson S K Wake J Bonner 《Biochemical and biophysical research communications》1974,58(1):50-57
The stoichiometry of rat liver chromatin proteins has been estimated by quantitative disc electrophoresis. There are about 1.3 × 108 histone and 2.4 × 107 nonhistone polypeptide molecules per haploid genome, an average of one such molecule for every 21 and 117 base pairs of DNA, respectively. Nonhistone proteins have been separated into 115 polypeptide size classes. The total number of molecules represented in distinct size classes ranges from 4.2 × 103 (limits of sensitivity) to 1.7 × 106 per haploid genome, with a mean of 2.1 × 105. 相似文献
50.
Catimel B; Scott AM; Lee FT; Hanai N; Ritter G; Welt S; Old LJ; Burgess AW; Nice EC 《Glycobiology》1998,8(9):927-938
We describe a novel immobilization technique to investigate interactions
between immobilized gangliosides (GD3, GM1, and GM2) and their respective
antibodies, antibody fragments, or binding partners using an optical
biosensor. Immobilization was performed by direct injection onto a
carboxymethyldextran sensor chip and did not require derivatization of the
sensor surface or the ganglioside. The ganglioside appeared to bind to the
sensor surface by hydrophobic interaction, leaving the carbohydrate epitope
available for antibody or, in the case of GM1, cholera toxin binding. The
carboxyl group of the dextran chains on the sensor surface did not appear
to be involved in the immobilization as evidenced by equivalent levels of
immobilization following conversion of the carboxyl groups into acyl amino
esters, but rather the dextran layer provided a hydrophilic coverage of the
sensor chip which was essential to prevent nonspecific binding. This
technique gave better reactivity and specificity for anti- ganglioside
monoclonal antibodies (anti-GD3: KM871, KM641, R24; and anti-GM2: KM966)
than immobilization by hydrophobic interaction onto a gold sensor surface
or photoactivated cross-linking onto carboxymethydextran. This rapid
immobilization procedure has facilitated detailed kinetic analysis of
ganglioside/antibody interactions, with the surface remaining viable for a
large number of cycles (>125). Kinetic constants were determined from
the biosensor data using linear regression, nonlinear least squares and
equilibrium analysis. The values of kd, ka, and KAobtained by nonlinear
analysis (KAKM871 = 1.05, KM641 = 1.66, R24 = 0.14, and KM966 = 0.65 x
10(7) M- 1) were essentially independent of concentration and showed good
agreement with data obtained by other analytical methods.
相似文献