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31.
J M Girardot D O Mack R A Floyd B C Johnson 《Biochemical and biophysical research communications》1976,70(2):655-662
The protein carboxylating system derived from vitamin K-deficient rat liver microsomes functions in detergent solution if vitamin K1, NADH, dithiothreitol, CO2 and O2 are added. The requirements for added NADH, dithiothreitol and O2 are all eliminated by the use of vitamin K1 hydroquinone in place of quinone. The use of the hydroquinone gives a more rapid reaction and a higher yield than does the quinone plus reducing system. The reaction proceeding from either the vitamin K1 quinone or hydroquinone is blocked by the spin-trapping agent, 5,5-dimethyl-l-pyrroline-N-oxide, suggesting that the active form of vitamin K is the semiquinone. 相似文献
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Rapid and efficient site-directed mutagenesis by single-tube 'megaprimer' PCR method. 总被引:24,自引:1,他引:24 下载免费PDF全文
We describe a rapid and efficient megaprimer PCR procedure for site-directed mutagenesis that does not require any intermediate purification of DNA between the two rounds of PCR. This protocol is based on the design of forward and reverse flanking primers with significantly different melting temperatures ( T m). A megaprimer is synthesized in the first PCR reaction using a mutagenic primer, the low T m flanking primer and a low annealing temperature. The second PCR reaction is performed in the same tube as the first PCR and utilizes the high T m flanking primer, the megaprimer product of the first PCR and a high annealing temperature, which prevents priming by the low T m primer from the first PCR reaction. We have used this protocol with two different plasmids to produce cDNAs encoding seven distinct mutated proteins. We have observed an average mutagenesis efficiency of 82% in these experiments. 相似文献
33.
Selective precipitation and purification of monovalent proteins using oligovalent ligands and ammonium sulfate 总被引:1,自引:0,他引:1
Mirica KA Lockett MR Snyder PW Shapiro ND Mack ET Nam S Whitesides GM 《Bioconjugate chemistry》2012,23(2):293-299
This paper describes a method for the selective precipitation and purification of a monovalent protein (carbonic anhydrase is used as a demonstration) from cellular lysate using ammonium sulfate and oligovalent ligands. The oligovalent ligands induce the formation of protein-ligand aggregates, and at an appropriate concentration of dissolved ammonium sulfate, these complexes precipitate. The purification involves three steps: (i) the removal of high-molecular-weight impurities through the addition of ammonium sulfate to the crude cell lysate; (ii) the introduction of an oligovalent ligand and the selective precipitation of the target protein-ligand aggregates from solution; and (iii) the removal of the oligovalent ligand from the precipitate by dialysis to release the target protein. The increase of mass and volume of the proteins upon aggregate formation reduces their solubility, and results in the selective precipitation of these aggregates. We recovered human carbonic anhydrase, from crude cellular lysate, in 82% yield and 95% purity with a trivalent benzene sulfonamide ligand. This method provides a chromatography-free strategy of purifying monovalent proteins--for which appropriate oligovalent ligands can be synthesized--and combines the selectivity of affinity-based purification with the convenience of salt-induced precipitation. 相似文献
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Inter-patient variation in efficacy of five oncolytic adenovirus candidates for ovarian cancer therapy 总被引:2,自引:0,他引:2
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Mansour S. Alturki Ngolui Rene Fuanta Madison A. Jarrard Judith V. Hobrath Douglas C. Goodwin Thankhoe A. Rantso Angela I. Calderón 《Bioorganic & medicinal chemistry letters》2018,28(4):802-808
Single dose high-throughput screening (HTS) followed by dose-response evaluations is a common strategy for the identification of initial hits for further development. Early identification and exclusion of false positives is a cost-saving and essential step in early drug discovery. One of the mechanisms of false positive compounds is the formation of aggregates in assays. This study evaluates the mechanism(s) of inhibition of a set of 14 compounds identified previously as actives in Mycobacterium tuberculosis (Mt) cell culture screening and in vitro actives in Mt shikimate kinase (MtSK) assay. Aggregation of hit compounds was characterized using multiple experimental methods, LC-MS, 1HNMR, dynamic light scattering (DLS), transmission electron microscopy (TEM), and visual inspection after centrifugation for orthogonal confirmation. Our results suggest that the investigated compounds containing oxadiazole-amide and aminobenzothiazole moieties are false positive hits and non-specific inhibitors of MtSK through aggregate formation. 相似文献
37.
Qiu Y Patwa TH Xu L Shedden K Misek DE Tuck M Jin G Ruffin MT Turgeon DK Synal S Bresalier R Marcon N Brenner DE Lubman DM 《Journal of proteome research》2008,7(4):1693-1703
Colorectal cancer (CRC) remains a major worldwide cause of cancer-related morbidity and mortality largely due to the insidious onset of the disease. The current clinical procedures utilized for disease diagnosis are invasive, unpleasant, and inconvenient; hence, the need for simple blood tests that could be used for the early detection of CRC. In this work, we have developed methods for glycoproteomics analysis to identify plasma markers with utility to assist in the detection of colorectal cancer (CRC). Following immunodepletion of the most abundant plasma proteins, the plasma N -linked glycoproteins were enriched using lectin affinity chromatography and subsequently further separated by nonporous silica reversed-phase (NPS-RP)-HPLC. Individual RP-HPLC fractions were printed on nitrocellulose coated slides which were then probed with lectins to determine glycan patterns in plasma samples from 9 normal, 5 adenoma, and 6 colorectal cancer patients. Statistical tools, including principal component analysis, hierarchical clustering, and Z-statistics analysis, were employed to identify distinctive glycosylation patterns. Patients diagnosed with colorectal cancer or adenomas were shown to have dramatically higher levels of sialylation and fucosylation as compared to normal controls. Plasma glycoproteins with aberrant glycosylation were identified by nano-LC-MS/MS, while a lectin blotting methodology was used to validate proteins with significantly altered glycosylation as a function of cancer progression. The potential markers identified in this study for diagnosis to distinguish colorectal cancer from adenoma and normal include elevated sialylation and fucosylation in complement C3, histidine-rich glycoprotein, and kininogen-1. These potential markers of colorectal cancer were subsequently validated by lectin blotting in an independent set of plasma samples obtained from 10 CRC patients, 10 patients with adenomas, and 10 normal subjects. These results demonstrate the utility of this strategy for the identification of N -linked glycan patterns as potential markers of CRC in human plasma, and may have the utility to distinguish different disease states. 相似文献
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Vincent Madison 《Biopolymers》1985,24(1):97-103
A multistep computational scheme was used to deduce possible conformations for a cyclic antagonist analog of somatostatin that has been reported by Coy and coworkers. An algebraic algorithm was used to find dihedral angles that give cyclic structures, the energy was computed for these structures, the lower-energy structures were classified into conformational families, the energy was minimized for the lowest-energy member of each family, and finally, the structures from energy minimization were reclassified. Analysis revealed seven distinct conformational families that contain reverse turns. The families differ in the position of the turns in the primary sequence; frame-shifted turns are observed at each possible position. 相似文献