全文获取类型
收费全文 | 4800篇 |
免费 | 383篇 |
国内免费 | 1013篇 |
出版年
2024年 | 17篇 |
2023年 | 112篇 |
2022年 | 120篇 |
2021年 | 153篇 |
2020年 | 182篇 |
2019年 | 198篇 |
2018年 | 161篇 |
2017年 | 180篇 |
2016年 | 188篇 |
2015年 | 165篇 |
2014年 | 237篇 |
2013年 | 407篇 |
2012年 | 238篇 |
2011年 | 278篇 |
2010年 | 202篇 |
2009年 | 285篇 |
2008年 | 240篇 |
2007年 | 255篇 |
2006年 | 272篇 |
2005年 | 260篇 |
2004年 | 236篇 |
2003年 | 205篇 |
2002年 | 200篇 |
2001年 | 157篇 |
2000年 | 138篇 |
1999年 | 112篇 |
1998年 | 94篇 |
1997年 | 78篇 |
1996年 | 78篇 |
1995年 | 83篇 |
1994年 | 75篇 |
1993年 | 50篇 |
1992年 | 40篇 |
1991年 | 40篇 |
1990年 | 58篇 |
1989年 | 31篇 |
1988年 | 27篇 |
1987年 | 31篇 |
1986年 | 28篇 |
1985年 | 46篇 |
1984年 | 62篇 |
1983年 | 33篇 |
1982年 | 20篇 |
1981年 | 23篇 |
1980年 | 22篇 |
1979年 | 22篇 |
1978年 | 16篇 |
1977年 | 7篇 |
1976年 | 16篇 |
1974年 | 8篇 |
排序方式: 共有6196条查询结果,搜索用时 921 毫秒
11.
Pavel Veprek Jan Jezek Jirí Velek Hatem Tallima Mona Montash Rashika El Ridi 《Journal of peptide science》2004,10(6):350-362
Four monoepitopic MAPs (MAP A, B, C and E) and one bis-diepitopic MAP B-E derived fromthe primary sequence of Schistosoma mansoni glyceraldehyde 3-phosphate dehydrogenase, previously tested in BALB/c mice, were examined for their immunogenicity and protective capacity in C57BL/6 mice. Despite multimerization into MAPs, MAP Aand MAP C were poorly immunogenic. In contrast toBALB/c mice, MAP E was non-immunogenic in C57BL/6 mice. Peptide B in the form of MAP B orbis-diepitopic MAPB-E elicited immune responses in C57BL/6 mice that were associated with a significant decrease in worm burden. The MAPs were prepared by the stepwise solid-phase peptide synthesis using Boc/Bzl chemistry, successfully purified on the RP-HPLC column and characterized by RP-HPLC, HPCE and MALDI-TOF MS techniques. A general strategy for MAPs purification is discussed here and the purification of MAP Band MAP E is documented in detail. 相似文献
12.
Summary A method for isolating high quality DNA from wholeEuglena cells is described. The procedure consists in: the weakening of the cell pellicle in glycerol avoiding the mechanical disruption of cells and shearing damage in DNA molecules; the decondensation ofEuglena compact chromatin directly inside the cells; the complete dissociation of cells and nucleoproteins in sarkosyl detergent; the optional digestion of proteins and RNA with DNase-free enzymes and the final purification of DNA by isopycnic banding in CsCl gradients. Degradation of DNA is prevented all along the extraction procedure by glycerol, antioxydants, EDTA and sarkosyl detergent. Using the enzymatic digestion step, DNA containing few single-stranded nicks is obtained with a yield approaching 100%. DNA with no single-stranded nick could be obtained with a 35% yield when the enzymatic digestion step was omitted. In both cases, the double-stranded DNA has an average molecular weight equal or greater than 6×107. It is free of contaminants and could be easily digested with restriction enzymes. After digestion with Eco RI and size-fractionation in agarose gel this DNA has permitted specific hybridization of the rDNA sequences with a radioactive rRNA probe.Abbreviations Kbp
kilobasepairs
- Kb
kilobases 相似文献
13.
N-Terminal Sequence of Pig Brain Choline Acetyltransferase Purified by a Rapid Procedure 总被引:6,自引:5,他引:1
Axel Braun Yves-Alain Barde Friedrich Lottspeich Werner Mewes Hans Thoenen 《Journal of neurochemistry》1987,48(1):16-21
A procedure is reported that allows the purification and amino terminal sequencing of pig brain choline acetyltransferase. The enzyme (present in extremely low amounts in this tissue) is eluted together with its antibody from an affinity column by a mild pH shift and the resulting enzyme-antibody complex separated by gel electrophoresis. The band corresponding to the enzyme is electroeluted from the gel using volatile solutions allowing the direct determination of the amino acid composition and partial sequence. The first 11 residues are: Pro-Ile-Leu-Glu-Lys-Thr-Pro-Pro-Lys-Met-Ala. 相似文献
14.
Ferredoxin-NADP reductase from Euglena gracilis Klebs var. Bacillaris Cori purified to apparent homogeneity, yields a typical 36 kDa and an unusual 15 kDa polypeptide on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, exhibits a typical flavoprotein spectrum, contains FAD, and catalyzes NADPH-dependent iodonitrotetrazolium-violet diaphorase, NADPH-specific ferredoxin-dependent cytochrome-c-550 reductase and NADPH-NAD transhydrogenase activities. Rabbit antibody to the purified FNR blocks these activities specifically and also blocks the iodonitrotetrazolium-violet diaphorase activity of Euglena chloroplasts completely. The low iodonitrotetrazolium-violet diaphorase activity in the plastidless mutant, W10BSmL, is mitochondrial and is not specifically blocked by the ferredoxin-NADP reductase antibody. Dark-grown non-dividing (resting) wild-type Euglena cells show a 4-fold increase in ferredoxin-NADP reductase activity during greening at 970 lx. Half of the low ferredoxin-NADP reductase activity in dark-grown cells is initially soluble, but by the end of chloroplast development nearly all of the enzyme is membrane-bound. The binding of ferredoxin-NADP reductase on exposure to light correlates with the extent of thylakoid membrane formation. Immunoblots of wild-type extracts during greening indicate that the 15 kDa polypeptide increases in the same manner as the extent of reductase binding to thylakoid membranes. 相似文献
15.
Glutamine synthetase (GS) from the chick brain was purified to apparent homogeneity by ammonium sulfate fractionation followed by affinity chromatography, electrofocusing and Sephadex G-150 chromatography. The purified enzyme showed a single band on sodium dodecyl sulfate analysis in polyacrylamide gel. By sedimentation equilibrium analysis and gel electrophoresis analysis, it was shown that the enzyme has a subunit molecular weight of 45,000 and a native molecular weight of 364,000, which is consistent with an octameric structure. Sedimentation analysis in the presence of Mg2+ revealed three different forms of macromolecules corresponding respectively to a monomer, a tetramer and an octamer. Among eight cations tested (Ca2+, Co2+, Fe2+, Li+, Mg2+, Mn2+, Ni2+, Zn2+) only Co2+, Mg2+ and Mn2+ supported GS activity; the order of activatory ability was Mg2+>Co2+>Mn2+. The maximum activating effect of Mn2+ occurs only within a very narrow range of concentration: with an excess of cation causing strong inhibition of GS activity. For each cation, maximal GS activity occurs at a defined cation/ATP ratio. A regulatory system in which Mn2+, modulates the Mg2+ dependent GS activity, is proposed; such cation interactions may be of significance in the intracellular control of glutamine synthesis. 相似文献
16.
The engineering considerations common to large scale chromatographic purification of proteins are reviewed. A discussion of the industrial chromatography fundamentals is followed by aspects which affect the scale of separation. The separation column geometry, the effect of the main operational parameters on separation performance, and the physical characteristics of column packing are treated. Throughout, the emphasis is on ion exchange and size exclusion techniques which together constitute the major portion of commercial chromatographic protein purifications. In all cases, the state of current technology is examined and areas in need of further development are noted.
The physico-chemical advances now underway in chromatographic separation of biopolymers would ensure a substantially enhanced role for these techniques in industrial production of products of new biotechnology. 相似文献
17.
Summary The adult rat lung cytoplasm contains some factors which markedly stimulate adenylate cyclase activity in plasma membranes (Nijjar, M. S. Biochim. Biophys. Acta 584:43–50, 1979). Adenylate cyclase activator (ACA) was purified from rat lungs by DEAE-cellulose chromatography, preparative isoelectric focusing and by repeated high-performance liquid chromatography on a Sepharogel TSK 2000SW column. The final preparation showed about 200 fold purification in ACA activity over the original lung supernatant, and appeared to be homogeneous on the basis of its migration into a single band on SDS-polyacrylamide gel electrophoresis, and co-elution of ACA activity with protein from a gel exclusion column. ACA is an acidic (pl 4.8 ± 0.1), heat labile, monomeric protein of 40000 ± 2000 dalton molecular weight, and does not resemble calmodulin. 相似文献
18.
H. G. Aghajanyan A. M. Arzumanyan A. A. Arutunyan T. N. Akopyan 《Neurochemical research》1988,13(8):721-727
Two cystatins were purified from tissue extract of bovine brain by alkaline treatment, acetone fractionation, gel chromatography on Sephadex G-75, and affinity chromatography on S-carboxymethyl-papain-Sepharose. One of the inhibitors had a relatively high molecular mass, 25 kDa (HMM-cystatin) with pI 4.7, and the other, 11 kDa (LMM-cystatin) with pI 5.23. Both inhibitors showed considerable stability at pH 2 and 80°C. The cystatins inhibited papain, ficin, and cathepsins B and H, but not trypsin, chymotrypsin, thermolysin, nagarse, and cathepsin D. Ki values for the complexes of papain and the inhibitors were estimated to be 2.8×10–10 M for HMM-cystatin and 1.3×10–9 M for LMM-cystatin. Both purified cystatins prevented degradation of substance P by soluble fraction and lysosomal extract obtained from synaptosomes, but did not suppress the cleavage of the peptide by synaptosomal plasma membranes.Abbreviations HMM-cystatin
high molecular mass inhibitor
- LMM-cystatin
low molecular mass inhibitor
- SP
substance P
- SPM
synaptosomal plasma membranes
- p-CMB
4-chloromercuribenzoic acid
- BK
bradykinin
- Bz-Arg-Nap
N-benzoyl-dl-arginine--naphthylamide
- Arg-Nap
dl-arginine--naphthylamide
- P-Pxy-Hb
hemoglobin initially coupled with pyridoxal-5-phosphate 相似文献
19.
Ira D. Bhattacharya M. F. Picciano John A. Milner 《Biological trace element research》1988,18(1):59-70
Human milk glutathione peroxidase (GPx) was purified 4500-fold using acetone precipitation and purification by repetitive
ion-exchange and gel filtration chromatography with an overall yield of 34%. Homogeneity was established by gel electrophoresis.
Using gel filtration, the molecular weight (mol wt) of the enzyme was estimated to be 92 kdalton (kD). The monomeric molecular
weight was estimated to b 23 kD from polyacrylamide gel electrophoresis, indicating that the native enzyme consists of four
identical subunits. The molecular weight of each subunit was supported by amino acid analysis. Selenium (Se) content of the
purified enzyme was 0.31%, in a stoichiometry of 3.7 g-atoms/mol. Data from these studies reveal that GPx provided approximately
22% of total milk Se, but only 0.025% of the total protein. 相似文献
20.
Coenzyme A-linked acetaldehyde dehydrogenase (ACDH) of ethanol-grown cells of Acetobacterium woodii was purified to apparent homogeneity; a 28-fold purification was achieved with 13% yield. The enzyme proved to be oxygen-sensitive and was inactive in the absence of dithioerythritol. During the purification procedure addition of 1 mM MgCl2 was necessary to maintain enzyme activity. Alcohol dehydrogenase (ADH) activity was separated from ACDH during anion exchange chromatography using DEAE Sephacel. A part of the ACDH activity coeluted with ADH, but both could be separately eluted from a Cibacron Blue 3GA-Agarose column, revealing the same subunit structure and activity band for ACDH as found before and, thus, indicating an aggregation of the enzyme. The remaining ADH activity could be separated by gel filtration. For the native ACDH a molecular mass of 255 kDa was determined by polyacrylamide gel electrophoresis and of 272 kDa by gel filtration using Superose 12. The enzyme subunit sizes were 28 kDa and 40 kDa, respectively, indicating a 44 structure for the active form. The enzyme catalyzed the oxidation of several straight chain aldehydes although it was most active with acetaldehyde. NADH strongly inhibited oxidation of acetaldehyde whereas NADPH had no effect. The inhibition was noncompetitive.Non-standard abbrevations ACDH
acetaldehyde dehydrogenase
- ADH
alcohol dehydrogenase
- CHES
2-(N-cyclohexylamino)-ethanesulfonate
- DTE
dithioerythritol
- KP-buffer
25 mM K-PO4, pH 7.5, containing, 4 mM DTE
- MES
2-(N-morpholino)-ethanesulfonate
- TAPS
N-Tris-(hydroxymethyl)-methyl-3-aminopropa-nesulfonate 相似文献