全文获取类型
收费全文 | 6246篇 |
免费 | 302篇 |
国内免费 | 311篇 |
出版年
2023年 | 86篇 |
2022年 | 93篇 |
2021年 | 75篇 |
2020年 | 111篇 |
2019年 | 143篇 |
2018年 | 187篇 |
2017年 | 113篇 |
2016年 | 130篇 |
2015年 | 140篇 |
2014年 | 311篇 |
2013年 | 399篇 |
2012年 | 179篇 |
2011年 | 355篇 |
2010年 | 322篇 |
2009年 | 316篇 |
2008年 | 283篇 |
2007年 | 288篇 |
2006年 | 296篇 |
2005年 | 297篇 |
2004年 | 223篇 |
2003年 | 173篇 |
2002年 | 137篇 |
2001年 | 87篇 |
2000年 | 86篇 |
1999年 | 107篇 |
1998年 | 85篇 |
1997年 | 94篇 |
1996年 | 52篇 |
1995年 | 88篇 |
1994年 | 93篇 |
1993年 | 74篇 |
1992年 | 74篇 |
1991年 | 63篇 |
1990年 | 47篇 |
1989年 | 50篇 |
1988年 | 38篇 |
1987年 | 46篇 |
1986年 | 31篇 |
1985年 | 111篇 |
1984年 | 260篇 |
1983年 | 159篇 |
1982年 | 156篇 |
1981年 | 98篇 |
1980年 | 88篇 |
1979年 | 78篇 |
1978年 | 50篇 |
1977年 | 26篇 |
1976年 | 16篇 |
1975年 | 12篇 |
1974年 | 20篇 |
排序方式: 共有6859条查询结果,搜索用时 15 毫秒
101.
The localization of the dissimilatory sulfite reductase in Desulfovibrio desulfuricans strain Essex 6 was investigated. After treatment of the cells with lysozyme, 90% of the sulfite reductase activity was found in the membrane fraction, compared to 30% after cell rupture with the French press. Sulfite reductase was purified from the membrane (mSiR) and the soluble (sSiR) fractiion. On SDS-PAGE, both mSiR and sSiR exhibited three bands at 50, 45 and 11 kDa, respectively. From their UV/VIS properties (distinct absorption maxima at 391, 410, 583, 630 nm, enzymes as isolated) and the characteristic red fluorescence in alkaline solution, mSiR and sSiR were identified as desulfoviridin. Sulfite reductase (HSO3
-H2S) activity was reconstituted by coupling of mSiR to hydrogenase and cytochrome c
3 from D. desulfuricans. The specific activity of mSiR was 103 nmol H2 min-1 mg-1, and sulfide was the major product (72% of theoretical yield). No coupling was found with sSiR under these conditions. Furthermore, carbon monoxide was used to diferentiate between the membrane-bound and the soluble sulfite reductase. In a colorimetric assay, with photochemically reduced methyl viologen as redox mediator, CO stimulated the activity of sSiR significantly. CO had no effect in the case of mSiR. These studies documented that, as isolated, both forms of sulfite reductase behaved differently in vitro. Clearly, in D. desulfuricans, the six electron conversion HSO3
-H2S was achieved by a membranebound desulfoviridin without the assistance of artificial redox mediators, such as methyl viologen.Abbreviations
SiR
sulfite reductase
-
mSiR
sulfite reductase purified from membranes
-
sSiR
sulfite reductase purified from the soluble fraction
Enzymes Sulfite reductase, EC 1.8.99.1 Cytochrome c
3 hydrogenase, EC 1.12.2.1 相似文献
102.
Interaction of phlorizin, a potent inhibitor of the Na+/D-glucose cotransporter, with the NADPH-binding site of mammalian catalases. 下载免费PDF全文
T. Kitlar F. Dring D. F. Diedrich R. Frank H. Wallmeier R. K. Kinne J. Deutscher 《Protein science : a publication of the Protein Society》1994,3(4):696-700
Phlorizin is a reversible inhibitor of the renal and small intestinal Na+/D-glucose cotransporter. In an attempt to purify the Na+/D-glucose cotransporter from a pig kidney brush border membrane fraction, we used an Affi-Gel affinity chromatography column to which 3-aminophlorizin had been coupled. A protein, composed according to crosslinking experiments of at least 3 subunits of molecular weight 60 kDa, was found to bind specifically to the phlorizin column. This protein was subsequently identified as catalase by sequence homology of three of its tryptic fragments to the sequence of several mammalian catalases as well as by its enzymatic activity. Although bovine liver catalase was bound tightly to the affinity matrix, phlorizin had no effect on the ability of the enzyme to degrade H2O2. In contrast, the Aspergillus niger and Neurospora crassa catalases did not bind to the phlorizin column. This difference may be related to the fact that mammalian catalases, but not the fungal catalases, contain an NADPH binding site with a yet unknown function. Interestingly, bovine liver catalase could be eluted with 50 microM NADPH from phlorizin columns. Irradiation in the presence of [3H]4-azidophlorizin allowed photolabeling of bovine liver catalase, which was prevented by the presence of 10 microM NADPH. After digestion of photolabeled catalase with chymotrypsin, a radioactive peptide was detected that was absent in catalase protected with NADPH. Docking simulations suggested that phlorizin can bind to the NADPH binding site with high affinity. 相似文献
103.
By computer simulation of experimental dynamic gas chromatographic elution profiles, the rotational energy barrier ΔG= of racemic 2,2′-diisopropylbiphenyl has been determined as 114.6–115.0 kJ/mol (75–100°C). These data are in good agreement with a value that was determined previously by measuring the racemization kinetics of an enriched sample. This indicates that there is no measurable catalytic or inhibitory effect of the stationary phase. © 1994 Wiley-Liss, Inc. 相似文献
104.
A new covalently-bonded, silica-based stationary phase, using as the chiral selector the 1-(3-aminopropyl) derivative of (+)-(5R,8S,10R)-terguride, has been developed to resolve optically active isomers by HPLC. Good resolution of structurally related racemic ergot alkaloids were obtained using water-methanol mixtures as the eluent. Analysis of the influence of the type and concentration of the organic modifier, and the pH of the buffer in the mobile phase allowed the enantioseparation of these compounds to be optimized. Determination of the optical purity of a lisuride-containig drug is reported. © 1994 Wiley-Liss, Inc. 相似文献
105.
Lifibrol, a new drug for the treatment of hypercholesterolemia, contains a stereogenic center bearing a secondary alcohol group. A normal-phase achiral–chiral HPLC separation of the enantiomers of lifibrol and two of its metabolites was developed and validated for quantitation in dog plasma. A silica and a Chiralcel OD-H column were operated in series and all six enantiomeric components and internal standard were directly separated. An initial solid-phase extraction (phenyl) clean-up step and a column-switching step to eliminate late-eluting compounds were also utilized. The solid-phase extraction step was automated using a robotic system. Assay development, validation, and application of the method to a bioavailability study of the racemate and enantiomers of lifibrol in dogs are described. The lower limit of quantitation was 0.0125 μg/ml for each enantiomer of lifibrol using 200 μl of dog plasma with UV detection (255 nm). In dog plasma following oral or intravenous administration of the racemate, the (R)/(S) ratio of the enantiomers of lifibrol was greater than one and increased with time. Following administration of the individual enantiomers, chiral inversion of the (S)-enantiomer but not the (R)-enantiomer was observed. © 1994 Wiley-Liss, Inc. 相似文献
106.
Production of mouse monoclonal antibodies using a continuous cell culture fermenter and protein G affinity chromatography 总被引:2,自引:0,他引:2
A. Zamboni I. Giuntini D. Gianesello F. Maddalena F. Rognoni D. Herbst 《Cytotechnology》1994,16(2):79-87
The production of anti--fetoprotein monoclonal antibodies for diagnostic use was carried out in a stirred tank fermenter equipped with a double membrane stirrer for bubble free aeration and continuous medium perfusion. A serum-free medium supplemented with 4 mM L-glutamine and 2.0 g/l glucose with a protein content of only 780 g/ml was used for the production process. The harvested antibodies were concentrated 50-fold using a tangential ultrafiltration system and were then purified in a one step purification process by protein G affinity chromatography. The purity of the final product (90%) was controlled by SDS-polyacrylamide gel electrophoresis, gel exclusion chromatography and isoelectric focussing. For further quality controls of the product the immunoglobulin subclass and the isoelectric point were determined and the specificity of the purified mAb was tested by RIA using125I labelled -fetoprotein.1.87 g of purified monoclonal antibodies were produced (90% purity) within 2 weeks. It was found that the use of this type of stirred tank fermenter combined with a one step purification process using protein G affinity chromatography represents a suitable method for the fast production of medium scale quantities (500 mg–5 g) of monoclonal antibodies for diagnostic use.Abbreviations AFP
-Fetoprotein
- BSA
bovine serum albumine
- FCS
Fetal calf serum
- HRP
horseradish peroxidase
- OPD
o-phenylenediamine dihydrochloride
- I.P.
isoelectric point
- IEF
isoelectric focussing
- PBS
Phosphate buffered saline 相似文献
107.
沈阳西部污灌水中有机污染物的分析刘海玲,张丽珊,姚家彪,于殿臣,朱岩,可夫,姜萍(中国科学院沈阳应用生态研究所,110015)AnalysisofOrganicPollutantsinIrrigatedsewageInWesternShenyang.... 相似文献
108.
The expression of smooth muscle myosin light chain kinase (MLCK) was investigated during chicken gizzard development. The molecular weight and the antigenic properties of MLCK did not change during development. The use of anion exchange high performance liquid chromatography (HPLC) enabled us to distinguish between MLCKs from post-hatched and adult chickens. A partial amino acid sequence determination of 4-day-old gizzard MLCK failed to disclose differences in the primary sequences of the two proteins. The results suggest that MLCK has the same primary sequence in all sequences of the two proteins. The results suggest that MLCK has the same primary sequence in all stages of gizzard development, although charge variants due to post-translational modifications may exist. 相似文献
109.
本文合成了一种腺苷亲和层析凝胶,并采用亲和层析法从牛脑细胞膜上分离出了几种膜上结合的腺苷结合蛋白质。这些蛋白质在SDS-PAGE电泳凝胶上为单一或主要的蛋白带,分子量分别为64kd,45kd,35kd。腺苷转运体抑制剂潘生丁和NBMPR对64kd蛋白与^3h-腺苷的结合抑制作用远强于腺苷受体的激动剂NECA和R-PIA;这表明64kd蛋白为牛脑细胞膜上结合的腺苷转运体。 相似文献
110.
应用于预生物合成研究的核酸碱基分析方法何裕建,戚生初(中国科学院生物物理研究所,北京10O101)(北京大学技术物理系,北京10O871)关键词HPLC分析;核酸碱基;离子交换色谱在生命起源研究中,核酸大分子起源是化学进化的一个重要课题,众所周知,核... 相似文献