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81.
Isolation of pituitary fibroblast growth factor by fast protein liquid chromatography (FPLC): partial chemical and biological characterization 总被引:14,自引:0,他引:14
Denis Gospodarowicz Sharon Massoglia Jannie Cheng Ge-Ming Lui Peter Bhlen 《Journal of cellular physiology》1985,122(2):323-332
Bovine pituitary fibroblast growth factor has been purified 222,000-fold to homogeneity by a combination of differential salt extraction, gel filtration, and ion exchange chromatography on Mono S column. Pituitary FGF is a single-chain polypeptide with an apparent molecular mass of 15,800 and an isoelectric point of 9.6. It is highly active in triggering the proliferation of bovine and human vascular endothelial cell [half-maximal stimulation at 23-40 pg/ml (1.5-2.6 pM) and saturation between 140 and 280 pg/ml (9.3-18.6 pM)]. It displays a similar activity on bovine vascular smooth muscle cells, corneal endothelial cells, granulosa and adrenal cortex cells, and rabbit costal chondrocytes. 相似文献
82.
C Y Cheng N A Musto G L Gunsalus J Frick C W Bardin 《The Journal of biological chemistry》1985,260(9):5631-5640
To determine how the androgen binding protein in human testes (hABP) is related to the serum protein, testosterone-estradiol binding globulin (hTeBG), both proteins were isolated and compared. The hABP in extracts of human testes was composed of two molecular species based on concanavalin A (ConA)-Sepharose chromatography. Form I hABP did not interact with ConA while Form II hABP bound to ConA and eluted with alpha-methylmannoside. Form I and Form II hABP from five batches of testes were then purified approximately 30,500- and 30,000-fold to apparent homogeneity by high-performance liquid chromatography and compared with hTeBG isolated from human pregnancy serum. Fractionation of both forms of hABP and hTeBG by polyacrylamide gel electrophoresis in the absence of sodium dodecyl sulfate suggested that the native forms of these proteins were indistinguishable. However, analysis of the purified proteins on sodium dodecyl sulfate-containing polyacrylamide gels indicated that all three were dimers and that each was composed of monomers of at least two sizes which were not present in equimolar concentrations. Two distinctive monomers or protomers of each protein were designated as heavy (H) and light (L) according to their electrophoretic mobilities on sodium dodecyl sulfate-polyacrylamide gels. The H and L protomers of Form I hABP showed apparent molecular weights of 55,000 and 52,000, respectively, in all preparations and were usually present in a 4:5 ratio (H:L). The two components of Form II hABP had apparent molecular weights of 53,000 and 48,000, respectively, and existed in a ratio of approximately 20:1. These two components could not be distinguished in some preparations where Form II hABP migrated as a broad band rather than as distinct protomers. By contrast, hTeBG, which was similar to Form II hABP with respect to ConA binding, always exhibited discrete H and L protomers in a 10:1 ratio. Photolysis of these highly purified proteins with delta 6-[3H]testosterone resulted in specific covalent labeling of their binding sites, confirming that the products identified by silver staining and immunoblotting were indeed steroid binding proteins. The H and L protomers of Form I hABP and hTeBG were separated and examined by peptide mapping using Staphylococcus aureus protease V8 and chymotrypsin. The comparison of the respective fragmentation patterns of protomers indicated that Form I hABP and hTeBG contained distinctive peptides.(ABSTRACT TRUNCATED AT 400 WORDS) 相似文献
83.
Adenosine 5'-diphosphate as an allosteric effector of phosphorylase kinase from rabbit skeletal muscle 总被引:2,自引:0,他引:2
Equilibrium binding and activity studies indicate that adenosine 5'-diphosphate binds to phosphorylase kinase with high affinity at a site, or sites, distinct from the catalytic site. Equilibrium dialysis at pH 6.8 and 8.2, with and without Mg2+, and with phosphorylated and nonphosphorylated enzyme preparations revealed approximately 8 ADP binding sites per alpha 4 beta 4 gamma 4 delta 4 hexadecamer, with Kd values ranging from 0.26 to 17 microM. Decreasing the pH from 8.2 to 6.8 or removing the Mg2+ enhanced the affinity for ADP. At pH 6.8, ADP stimulated the phosphorylase conversion and autophosphorylation activities of the nonactivated enzyme. Analogs of ADP with modifications at the 2'-, 3'-, and 5'-positions allowed determination of structural requirements for the stimulation of activity. ADP seems to alter the conformation of the beta subunit because addition of the nucleotide inhibits its dephosphorylation by phosphoprotein phosphatase and its chemical cross-linking by 1,5-difluoro-2,4-dinitrobenzene. The binding affinities and effects of ADP suggest that it may function physiologically as an allosteric effector of phosphorylase kinase. 相似文献
84.
Human complement protein C8 was labeled with the fluorescent chromophores fluorescein-5-isothiocyanate (FITC), 3-(4-isothiocyanatophenyl)-7-diethylamine-4-methyl coumarin (IPM), eosin-5-isothiocyanate (EOS), or Texas Red (sulforhodamine-101-sulfonyl chloride; TR) with only minor reduction in the specific hemolytic activity of the protein. The distribution of C5b-8 complexes bound to sheep erythrocyte membranes was investigated by monitoring fluorescence resonance energy transfer (RET) between the following RET donor/acceptor pairs of labeled C8: FITC-C8/EOS-C8, IPM-C8/EOS-C8, and FITC-C8/TR-C8. On binding to membranes containing pre-formed C5b67 complexes, specific RET was detected for each of the donor/acceptor pairs of labeled C8 investigated. In contrast, no energy transfer was observed for these RET donor/acceptor pairs of labeled C8 incubated in the presence of control membranes or in membrane-free solution. On the basis of a consideration of the transfer efficiency that would be expected for donor/acceptor pairs of labeled C8 that were uniformly dispersed on the membrane surface, these results suggest that C5b-8 complexes are aggregated into polymeric clusters when membrane-bound. The efficiency of donor-C8 to acceptor-C8 RET--and the hemolytic activity of membrane-bound C5b-8 (in the absence of C9)--are both related to the surface density of membrane-bound C5b67, suggesting that the physical clustering of the membrane-inserted C5b-8 complex may be related to the expression of its cytolytic activity. 相似文献
85.
This paper deals with the role of light in the germination of akinetes of Anabaena azollae. The two maxima action spectra are situated at 385 and 615 nm and the stimulation of the germination process by photosynthate was confirmed. The photoreceptor absorbing at 385 nm was identified as a flavin and that at 615 nm as a phytochrome. A model is suggested for the mode of action of light in the germination of akinetes of blue-green algae.C. Tsui 相似文献
86.
The role of the carbohydrate moiety on the size heterogeneity and immunologic determinants of human testosterone-estradiol-binding globulin 总被引:1,自引:0,他引:1
Human testosterone-estradiol-binding globulin (hTeBG) has been purified to apparent homogeneity by several laboratories using procedures which, in most instances, were labor intensive. In this report, hTeBG was purified from pregnancy serum by a newly developed two step procedure involving sequential affinity chromatography and ion-exchange high performance liquid chromatography (ion-exchange HPLC). The purity of the final product was confirmed by silver stained SDS-polyacrylamide gel and reverse phase HPLC monitored at 206 nm. hTeBG purified by ion-exchange-HPLC maintained binding activity by Dextran coated charcoal (DCC) assay and size heterogeneity on SDS-polyacrylamide gels which were indistinguishable from those of the proteins purified by conventional chromatography. Removal of the carbohydrate moiety from the molecule by both enzymatic and chemical treatment reduced the apparent molecular size and eliminated lectin binding of hTeBG subunits. Deglycosylation did not, however, abolish or alter the distribution of the protomeric forms of this subunit. We conclude that hTeBG is a dimer whose monomer exhibits two protomeric forms which is not a result of carbohydrate heterogeneity. In addition, disialylated and deglycosylated hTeBG exhibited antigenic determinants identical to the native protein. 相似文献
87.
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90.
马立克氏病毒单克隆抗体的研究 总被引:1,自引:0,他引:1
获得了4株分泌马立克氏病毒(MDV)特异性单克隆抗体(McAb)的杂交瘤细胞:4BS10对MDV所有毒株呈阳性反应;4CN8 对MDV血清1,3型毒株发生反应;2BN90和4CN24只对MDV血清1型毒株有阳性反应。3个McAb属IgG1,1个为IgG2b,均不中和MDV,免疫扩散试验也无沉淀线。对禽白血病毒(ALV)无交叉反应。 以2BN90和辣根过氧化物酶、异硫氰酸荧光素的结合物进行直接酶联免疫吸附试验和直接荧光抗体试验,均获得成功。抗体滴度前者为1/51,200,后者为1/640。对ALV无交叉反应。 相似文献