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81.
The chitinase gene of Manduca sexta was cloned into the expression vector, pET-28a, and expressed in Escherichia coli BL21 (DE3) host cells. The protein product was expressed in inclusion bodies. After denaturation and renaturation procedures using a Ni2+-NTA affinity chromatography column, soluble chitinase was obtained. The authenticity of the renatured protein was confirmed by Western blotting. Polyclonal antibodies to the purified protein were raised in rabbits. The antibody reacted specifically with the expressed chitinase and was used to quantify its presence in transgenic cotton being developed to resist attack by various insects.Revisions requested 24 September 2004; Revisions received 18 November 2004 相似文献
82.
Summary. The new amino acid complex nanoparticles of bismuth and leucine can be prepared very easily by a room temperature solid–solid reaction. The various characterizations indicate that the formula of the complex is BiCl[(CH3)2CHCH2CHNH2COO]21.5H2O. The crystal structure of the solid complex belongs to monoclinic system with the lattice parameters: a = 1.6036 nm, b = 1.9903 nm, c = 2.1979 nm and β=108.06°. The new solid complex is nanoparticles with average size about 80 nm. 相似文献
83.
Lange BM Kirfel G Gestmann I Herzog V González C 《Histochemistry and cell biology》2005,124(3-4):325-334
Experimental approaches in Drosophila melanogaster over the last 20 years have played a fundamental role in elucidating the function, structure and molecular composition of
the centrosome. However, quantitative data on the structure and function of the Drosophila centrosome are still lacking. This study uses, for the first time, whole mount electron microscopy in combination with negative
staining on isolated centrosomes from the early Drosophila embryos to analyze its dimensions, structure and capacity to nucleate microtubules in vitro. We show that these organelles
are on average 0.75 μm in diameter and have abundant pericentriolar material which often appears fibrillar and with bulbous
protrusions. Corresponding to the abundant pericentriolar material, extensive microtubule nucleation occurs. Quantification
of the number of microtubules nucleated showed that 50–300 active nucleation sites are present. We examined via electron microscopy
immunogold labeling the distribution of γ-tubulin, CNN, Asp and the MPM-2 epitopes that are phosphorylated through Polo and
the Cdk1 kinase. The distribution of these proteins is homogeneous, with the MPM-2 epitopes exhibiting the highest density.
In contrast, centrosomal subdomains are identified using a centriole marker to relate centrosome size to the centriole number
by electron microscopy. In conclusion, we present a clear-cut technique assaying and quantifying the microtubule nucleation
capacity and antigen distribution complementing molecular studies on centrosome protein complexes, cell organelle assembly
and protein composition. 相似文献
84.
S基因在甘蓝EDFs上的高分辨率荧光原位杂交定位 总被引:1,自引:0,他引:1
植物细胞壁及细胞质的存在和植物染色体所具有的高浓缩特性,限制高效率原位杂交定位在植物细胞内的进行。针对小型染色体芸薹属植物采用常规方法DNA制备纤维的效果不佳的特点,新建制备方法:利用减数分裂前期的染色体为材料,在硅化的玻片上先后通过蛋白酶解和乙醇:乙酸(3:1)的适当处理,采用移动界而法制备EDFs。制备的EDFs比未经伸长处理的染色体在经向和横向方面分别取得较高程度的伸长与膨胀,长度可达到89-257μm,比相应地中期染色体增长30107倍,分辨率可达42.853.0kb。利用SRK和SCR两种探针同时在甘蓝粗线期染色体和EDFs上进行了原位杂交,首次鉴定了S基因座在其单倍体基因组中单拷贝性。在杂交信号检测中尽管未经过信号放大,但仍然可以观察到清晰的绿色信号;经荧光显微镜观察,在单一的EDF上发现两个相距1μm的SCR和SRK的信号点,由此得出局部分辨率为4kb的最高伸长度。 相似文献
85.
Bhasmas are unique Ayurvedic metallic preparations with herbal juices/fruits, known in the Indian subcontinent since the seventh
century BC and widely recommended for treatment of a variety of chronic ailments. Twenty bhasmas based on calcium, iron, zinc,
mercury, silver, potassium, arsenic, copper, tin, and gemstones were analyzed for up to 18 elements by instrumental neutron
activation analysis, including their C, H, N, and S contents. In addition to the major constituent element found at % level,
several other essential elements such as Na, K, Ca, Mg, V, Mn, Fe, Cu, and Zn have also been found in μg/g amounts and ultratrace
(ng/g) amounts of Au and Co. These seem to remain chelated with organic ligands derived from medicinal herbs. The bhasmas
are biologically produced nanoparticles and are taken along with milk, butter, honey, or ghee (a preparation from milk), thus,
this makes these elements easily assimilable, eliminating their harmful effects and enhancing their biocompatibility. Siddha
Makaradhwaja, a mercury preparation is found to be stoichiometrically HgS without any traces of any other element. Similarly,
Swet Parpati is stoichiometrically KNO3 but is found to have Mn, Cu, Zn, Na, P, and Cl as well. An attempt has been made to correlate the metallic contents with
their medicinal importance. Na and K, the two electrolytic elements, seem to be well correlated, although K/Na varies in a
wide range from 0.06 to 95, with specifically low values for Ca-, Fe-, and Zn-based hasmas. K/P also varies in a wide range
from 0.23 to 12, although for most bhasmas (n=12)., it is 2.3±1.2. Further, Fe/Mn is linearly correlated (r=0.96) with Fe in nine noniron bhasmas. 相似文献
86.
Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs through activation of their ion channels and neurotransmitter receptors, and process information in part through activity-dependent release of gliotransmitters. Furthermore, astrocytes constitute the main uptake system for glutamate, contribute to potassium spatial buffering, as well as to GABA clearance. These cells therefore constantly monitor synaptic activity, and are thereby sensitive indicators for alterations in synaptically-released glutamate, GABA and extracellular potassium levels. Additionally, alterations in astroglial uptake activity or buffering capacity can have severe effects on neuronal functions, and might be overlooked when characterizing physiopathological situations or knockout mice. Dual recording of neuronal and astroglial activities is therefore an important method to study alterations in synaptic strength associated to concomitant changes in astroglial uptake and buffering capacities. Here we describe how to prepare hippocampal slices, how to identify stratum radiatum astrocytes, and how to record simultaneously neuronal and astroglial electrophysiological responses. Furthermore, we describe how to isolate pharmacologically the synaptically-evoked astroglial currents. 相似文献
87.
Efforts to characterize proteins found in the outer membrane (OM) of Gram-negative bacteria have been steadily increasing due to the promise of expanding our understanding of fundamental bacterial processes such as cell adhesion or cell wall biogenesis as well as the promise of finding potential vaccine- or drug-targets for virulent bacteria. We have developed a mass spectrometry-compatible experimental strategy that resulted in increased coverage of the OM proteome of a model organism, Caulobacter crescentus. The specificity of the OM enrichment step was improved by using detergent solubilization of the protein pellet, low-density cell culture conditions, and a surface-layer deficient cell line. Additionally, efficient gel-assisted digestion, high-resolution RP/RP-MS/MS, and rigorous bioinformatic analysis led to the identification of 234 proteins using strict identification criteria (≥ two unique peptides per protein; peptide false discovery rate <2%). Eighty-four of the detected proteins were predicted to localize to the OM or extracellular space. These results represent ~70% coverage of the predicted OM/extracellular proteome of C. crescentus. This analytical approach, which considers important experimental variables not previously explored in published OM protein studies, can be applied to other OM proteomic endeavors "as is" or with slight modification and should improve the large-scale study of this especially challenging subproteome. 相似文献
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