全文获取类型
收费全文 | 1742篇 |
免费 | 87篇 |
国内免费 | 80篇 |
出版年
2023年 | 29篇 |
2022年 | 40篇 |
2021年 | 37篇 |
2020年 | 36篇 |
2019年 | 33篇 |
2018年 | 44篇 |
2017年 | 40篇 |
2016年 | 50篇 |
2015年 | 37篇 |
2014年 | 81篇 |
2013年 | 114篇 |
2012年 | 62篇 |
2011年 | 87篇 |
2010年 | 65篇 |
2009年 | 92篇 |
2008年 | 92篇 |
2007年 | 84篇 |
2006年 | 88篇 |
2005年 | 59篇 |
2004年 | 62篇 |
2003年 | 67篇 |
2002年 | 56篇 |
2001年 | 41篇 |
2000年 | 36篇 |
1999年 | 34篇 |
1998年 | 24篇 |
1997年 | 22篇 |
1996年 | 24篇 |
1995年 | 29篇 |
1994年 | 36篇 |
1993年 | 26篇 |
1992年 | 25篇 |
1991年 | 15篇 |
1990年 | 16篇 |
1989年 | 21篇 |
1988年 | 15篇 |
1987年 | 22篇 |
1986年 | 23篇 |
1985年 | 18篇 |
1984年 | 31篇 |
1983年 | 16篇 |
1982年 | 24篇 |
1981年 | 13篇 |
1980年 | 19篇 |
1979年 | 15篇 |
1978年 | 2篇 |
1977年 | 2篇 |
1974年 | 2篇 |
1973年 | 1篇 |
1972年 | 2篇 |
排序方式: 共有1909条查询结果,搜索用时 15 毫秒
1.
The ability to metabolically label proteins with 35S-methionine is critical for the analysis of protein synthesis and turnover. Despite the importance of this approach, however, efficient labeling of proteins in vivo is often limited by a low number of available methionine residues, or by deleterious side-effects associated with protein overexpression. To overcome these limitations, we have created a methionine-rich variant of the widely used HA tag, called HAM, for use with ectopically expressed proteins. Here we describe the development of a series of vectors, and corresponding antisera, for the expression and detection of HAM-tagged proteins in mammalian cells. We show that the HAM tag dramatically improves the sensitivity of 35S-methionine labeling, and permits the analysis of Myc oncoprotein turnover even when HAM-tagged Myc is expressed at levels comparable to that of the endogenous protein. Because of the improved sensitivity provided by the HAM tag, the vectors and antisera described here should be useful for the analysis of protein synthesis and destruction at physiological levels of protein expression. 相似文献
2.
Fluorescent analysis of alpha-keto acids in serum and urine by high-performance liquid chromatography 总被引:11,自引:0,他引:11
A selective procedure using synthetic substrates for determination of exo-1,4,-beta-glucanases in a mixture of exoglucanases , endoglucanases , and beta-glucosidases is formulated. The heterobiosides , p- nithrophenyl -beta-D- cellobioside ( pNPC ) or p-nitrophenyl-beta-D-lactoside ( pNPL ), were used as selective substrates for the measurement of exoglucanase activity. The exoglucanases (especially cellobiohydrolases , which split off cellobiose units from the nonreducing end of the cellulose chain) specifically act on the agluconic bond (between p-nitrophenyl and the disaccharide moiety) and not on the holosidic bond (between the two glucose units of cellobiose). The interfering effect of beta-glucosidase, which acts on both agluconic and holosidic bonds, is overcome by the addition of D-glucono-1,5-delta-lactone, a specific inhibitor of beta-glucosidases. The interference of endoglucanases , which also act on both agluconic and holosidic bonds, can be compensated for by prior standardization of the assay procedure with a purified endoglucanase from the studied mixture of cellulases. 相似文献
3.
Benjamin S. Johnson Lexie Chafin Daniela Farkas Jessica Adair Ajit Elhance Laszlo Farkas Joseph S. Bednash James D. Londino 《Molecular & cellular proteomics : MCP》2022,21(7):100256
Identifying protein–protein and other proximal interactions is central to dissecting signaling and regulatory processes in cells. BioID is a proximity-dependent biotinylation method that uses an “abortive” biotin ligase to detect proximal interactions in cells in a highly reproducible manner. Recent advancements in proximity-dependent biotinylation tools have improved efficiency and timing of labeling, allowing for measurement of interactions on a cellular timescale. However, issues of size, stability, and background labeling of these constructs persist. Here we modified the structure of BioID2, derived from Aquifex aeolicus BirA, to create a smaller, highly active, biotin ligase that we named MicroID2. Truncation of the C terrminus of BioID2 and addition of mutations to alleviate blockage of biotin/ATP binding at the active site of BioID2 resulted in a smaller and highly active construct with lower background labeling. Several additional point mutations improved the function of our modified MicroID2 construct compared with BioID2 and other biotin ligases, including TurboID and miniTurbo. MicroID2 is the smallest biotin ligase reported so far (180 amino acids [AAs] for MicroID2 versus 257 AAs for miniTurbo and 338 AAs for TurboID), yet it demonstrates only slightly less labeling activity than TurboID and outperforms miniTurbo. MicroID2 also had lower background labeling than TurboID. For experiments where precise temporal control of labeling is essential, we in addition developed a MicroID2 mutant, termed lbMicroID2 (low background MicroID2), that has lower labeling efficiency but significantly reduced biotin scavenging compared with BioID2. Finally, we demonstrate utility of MicroID2 in mass spectrometry experiments by localizing MicroID2 constructs to subcellular organelles and measuring proximal interactions. 相似文献
4.
《Fungal biology》2020,124(2):83-90
Latterly, the upsurge in use of antifungal drugs has brought about the emergence of several drug-resistance strains, making it skeptical to continue relying on current therapeutic regime. In the necessity of resistance-free antifungal agent, flavonoids presented possibilities of replacing existing drugs, displaying antifungal activity against pathogenic fungi. Among them, quercetin, one of the most representative flavonoids, exhibited antifungal activity against Candida albicans. To inspect the further understanding regarding quercetin, the antifungal mode of action of quercetin was investigated. In the initial step, the apoptosis was monitored after quercetin treatment. Moreover, intracellular levels of Mg2+ was assessed and was determined that Mg2+ increase occurred under the influence of quercetin. In addition, several features of mitochondrial dysfunction were monitored. Mitochondrial dysfunction triggers decrease in mitochondrial redox levels and leads to disruption in mitochondrial antioxidant system. Increased intracellular ROS and decreased intracellular redox levels were also displayed, indicating the occurrence of overall disruption in antioxidant systems. Sequentially, DNA fragmentation was observed and this DNA damage in turn induces apoptosis. In analyses, hexaamminecobalt(III) chloride (Cohex) was applied to inhibit Mg2+ transport between cytosol and mitochondria. Cohex attenuated the effects induced by quercetin, which demonstrates that the presence of Mg2+ is essential in quercetin-induced apoptosis. 相似文献
5.
6.
Seed dispersal influences a wide range of ecological processes. However, measuring dispersal patterns, particularly long‐distance dispersal, has been a difficult task. Marking bird‐dispersed seeds with stable 15N isotopes has been shown to be a user‐friendly method to trace seed dispersal. In this study, we determined whether 15N urea solution could be used to enrich seeds of two common wind‐dispersed plants, Eupatorium glaucescens (Asteraceae) and Sericocarpus tortifolius (Asteraceae). We further tested if the water type (distilled versus tap) in 15N urea solutions influences the level and variability of enrichment of plant seeds, and if increasing spraying frequency per se increases enrichment. Because droughts may lower seed set or kill plants, we wanted to investigate if the additional use of an externally applied anti‐transpirant affects the intake of externally applied 15N into seeds. The results demonstrate that 15N enrichment of seeds can facilitate dispersal experiments with wind‐dispersed plants. The use of distilled water in 15N urea solutions did not increase 15N enrichment compared to tap water. Further, enrichment was more efficient at lower spray frequencies. Both the use of tap water and low frequencies could lower time, effort and project costs. The results suggest that species can be protected from drought using an anti‐transpirant without decreasing the incorporation of 15N into seeds. 相似文献
7.
Strategies for signal amplification in nucleic acid detection 总被引:3,自引:0,他引:3
S. Calin Andras J. Brian Power Edward C. Cocking Michael R. Davey 《Molecular biotechnology》2001,19(1):29-44
Many aspects of molecular genetics necessitate the detection of nucleic acid sequences. Current approaches involving target
amplification (in situ PCR, Primed in situ Labeling, Self-Sustained Sequence Replication, Strand Displacement Amplification), probe amplification (Ligase Chain Reaction,
Padlock Probes, Rolling Circle Amplification) and signal amplification (Tyramide Signal Amplification, Branched DNA Amplification)
are summarized in the present review, together with their advantages and limitations. 相似文献
8.
Gary L. Stiles Ruth H. Strasser Brian F. Kilpatrick Sabrina R. Taylor Robert J. Lefkowitz 《Biochimica et Biophysica Acta (BBA)/General Subjects》1984,802(3)
Photoaffinity labeling techniques have recently demonstrated that mammalian β1- and β2-adrenergic receptors reside on peptides of Mr 62 000–64 000. These receptor peptides are susceptible to endogenous metalloproteinases which produce peptides of Mr 30 000–55 000. Several proteinase inhibitors markedly attenuate this process, specifically EDTA and EGTA. In this study we investigated the functional significance of this proteolysis (and its inhibition) in the β2-adrenergic receptor-adenylate cyclase system derived from rat lung membranes. Membrane preparations containing proteolytically derived fragments of the receptor of Mr 40000–55 000 are fully functional with respect to their ability to bind β-adrenergic antagonist radioligands such as [3H]dihydroalprenolol and β-adrenergic antagonist photoaffinity reagents such as p-azido-m-[125I]iodobenzylcarazolol. They retain the ability to form a high-affinity, agonist-promoted, guanine nucleotide-sensitive complex thought to represent a ternary complex of agonist, receptor and guanine nucleotide regulatory protein. Nonetheless, after proteolysis, GTP is less able to revert this high-affinity receptor complex to one of lower affinity, and all aspects of adenylate cyclase stimulation are reduced. In addition, the functional integrity of the N protein in membranes prepared without proteinase inhibitors is reduced as assessed by reconstitution studies with the cyc[su− variant of S49 lymphoma cell membranes. These results suggest that endogenous proteolysis does not directly impair the ability of β-adrenergic receptors to either bind ligands or interact with the guanine nucleotide regulatory protein. However, they imply that endogenous proteolysis likely impairs the functionality of other components of the adenylate cyclase system, such as the nucleotide regulatory protein. 相似文献
9.
Graham V. Brown Ross L. Coppel Helena Vrbova Raelene J. Grumont Robin F. Anders 《Experimental parasitology》1982,53(2):279-284
Proteins of erythrocytic stages of Plasmodium falciparum were biosynthetically labeled at different times during the first cycle of in vitro synchronous cultivation after collection from patients in the Madang region of Papua New Guinea. Proteins were immunoprecipitated with a pool of hyperimmune serum collected in the region then analyzed by sodium dodecyl sulfate-gel electrophoresis. Antigens were recognized in all life cycle stages but the majority of antigens, particularly those of high molecular weight, were present in the mature forms of the parasite. 相似文献
10.
John H. Collins Bliss Forbush Lois K. Lane Eleanor Ling Arnold Schwartz Anita Zot 《生物化学与生物物理学报:生物膜》1982,686(1):7-12
Highly purified lamb kidney (Na++K+)-ATPase was photoaffinity labeled with the tritiated 2-nitro-5-azidobenzoyl derivative of ouabain (NAB-ouabain). The labeled (Na++K+)-ATPase was mixed with unlabeled carrier enzyme. Two proteolipid (γ1 and γ2) fractions were then isolated by chromatography on columns of Sepharose CL-6B and Sephadex LH-60. The two fractions were interchangeable when rechromatographed on the LH-60 column, suggesting that γ1 is an aggregated form of γ2. The total yield was 0.8–1.5 mol of γ component per mol of catalytic subunit recovered. This indicates that the γ component is present in stoichiometric amounts in the (Na++K+)-ATPase. The proteolipids that were labeled with NAB-ouabain copurified with the unlabeled proteolipids. 相似文献