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61.
Purification of a K(+)-channel protein of sarcoplasmic reticulum by assaying the channel activity in the planar lipid bilayer system 总被引:1,自引:0,他引:1
A K(+)-channel protein of the sarcoplasmic reticulum (SR) was purified by assaying the channel activity in a planar lipid bilayer system. The light fraction of SR vesicles was solubilized in 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) and fractionated by an anion-exchange chromatography and followed by gel filtration chromatography and affinity chromatography with concanavalin A. All fractions in each steps were mixed with asolectin solubilized in CHAPS and reconstituted into vesicles by dialysis. The channel activity of each fraction was assayed after the reconstituted vesicles had been fused into a planar lipid bilayer. The final fraction which showed the K(+)-channel activity contained only 100 kDa protein in a silver-stained gel after SDS-PAGE and an anti-Ca(2+)-ATPase antibody did not recognize the protein. The characteristics of the K(+)-channel were identical to those observed in native SR vesicles when using the same method. The channel showed a single-channel conductance of 120 pS in 0.1 M KCl and marked voltage dependence. The channel did not permeate Ca2+ and Cl- and was blocked by neomycin B. 相似文献
62.
Slalom chromatography, a size-dependent DNA fractionation method based on a new principle [Hirabayashi, J., & Kasai, K. (1989) Anal. Biochem. 178, 336-341], was systematically studied in detail. In this method, larger DNA fragments are eluted much later than smaller ones from columns packed with spherical microbeads. Elution of a series of DNA fragments was systematically examined by using columns packed with polymer-based packings of different diameter and different pore size for high-performance gel permeation chromatography. Packings of smaller diameter proved to be superior for resolving the smaller size range of DNA, while the reverse was the case for larger DNAs. Application of a faster flow rate led to larger retardation of every DNA fragment, while at the lowest flow rate applied (0.067 cm/min), all the fragments were eluted almost at the void volume. When the column temperature was lowered, retardation of DNA became larger. On the other hand, differences in the chemical nature and the pore size of packings, or in the hydrophobicity of the eluting solvent, had little effect on DNA retardation. Size-dependent fractionation of DNA was also achieved even on columns packed with nonporous packings having anionic groups (cation exchangers). In conclusion, these results confirmed the previous conclusion that slalom chromatography is not based on an adsorption or equilibrium phenomenon but should be attributed to a hydrodynamic phenomenon. 相似文献
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Takashi Muramatsu Hisako Muramatsu Masataka Kasai Sonoko Habu Ko Okumura 《Biochemical and biophysical research communications》1980,96(4):1547-1553
agglutinin (DBA), which is specific for terminal α-N-acetylgalactosamine, bound to a spontaneous leukemia cell of mice, but not to lymphoid cells of the host. The DBA receptors were isolated from the leukemia cell labeled with [3H]-galactose after detergent solubilization and affinity chromatography on DBA-agarose. The major component of the receptors migrated as a glycoprotein of apparent molecular weight 100,000 upon SDS gel electrophoresis. Alkaline treatment degraded the glycoproteins, releasing oligosaccharides of molecular weight around 1,000. 相似文献
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K Yokoyama J Mashimo N Kasai T Terao T Osawa 《Hoppe-Seyler's Zeitschrift für physiologische Chemie》1979,360(4):587-595
The membrane binding sites for lipopolysaccharide (LPS) were isolated by affinity chromatography of the solubilized membranes prepared from 125I-labeled mouse B-cells and T-cells on an affinity adsorbent prepared by coupling Salmonella minnesota R595 LPS to activated Sepharose 4B. The membrane proteins bound to the affinity adsorbent and eluted with 1.0% Triton X-100 were analyzed according to their mobility on polyacrylamide gel electrophoresis in sodium dodecylsulphate. These membrane proteins were further identified by immunoprecipitation with specific antisera. Immunoglobulins, possibly immunoglobulins M and D, were identified in the eluate from the B-cell membranes. The histocompatibility-2-complex proteins (H-2D, H-2K and Ia antigens) were also found to be binding sites for LPS on both B-cells and T-cells. 相似文献
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This report that (1) cells mediating NK activity in different inbred mouse strains selectively express one of two allelic products specified by theLy-5 locus (or a locus tightly linked to it) and (2) this surface structure may directly contribute to NK-mediated cytolysis, since Ly 5 antiserum specifically inhibits NK activity in vitro in the absence of complement. 相似文献
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l(+)-tartrate-[U-14C] or sucrose-[U-14C] was fed into grape berries and 14CO2 evolution was determined. 14CO2 evolution front l(+)-tartrate-[U-14C] was slightly higher in mature than immature berries, and that from sucrose-[U-14C] was higher in immature than mature ones. 14CO2 evolution from l(+)-tartrate-[U-14C] was irregular throughout the day until 2 or 3 weeks after flowering. This stage shifted to regular 14CO2 evolution until 6 or 7 weeks after flowering, and the mode of 14CO2 evolution showed diurnal variation; higher in the day than at night. Then the stage without variation of 14CO2 evolution followed 10 weeks after flowering. These observations indicate that tartrate is not biochemically inert in grape berries, while the amount of 14CO2 evolution from sucrose-[U-14C] was higher at night than in the day through the whole ripening process, except in the early stage. 相似文献