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11.
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
12.
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
13.
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
14.
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
15.
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
16.
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
17.
Françoise Farace Anne-Marie Le Ridant Bernard Escudier Thierry Hercend Frédéric Triebel 《Biotherapy》1992,5(1):11-20
We have attempted to improve negative selection procedures for the large scale purification of human CD
in3
–
CD56+ NK cells. In a series of experiments, purifications of NK cells from 108 PBMC were performed by T cell depletion using either direct or indirect anti-CD3 labeling and the Magnetic Activated Cell Separation (MACS) procedure. Contaminating CD3+ cells were still present using either one of these two different T cell depletion protocols as shown by phenotyping IL-2 supplemented cell cultures on day 12. A second cycle of purification was therefore added. When MACS and Dynabeads were compared as complementary procedures to the first MACS cycle starting with 108 cells, the Dynabeads method was found to be superior to the MACS with regard to the elimination of residual T cells. Starting from 109 PBMC, we showed that this MACS+Dynabeads procedure gave similar satisfactory results when compared to the scaling-up of a previously established two steps procedure using Dynabeads. These two approaches (MACS+Dynabeads and 2 cycles of Dynabeads) have been also tested in a clinical setting to purify NK cells from cancer patients prior toin vitro expansion. The results indicate that the two methods are equivalent with respect to purity and recovery rate; a slight advantage in terms of feasibility was found in favor of 2 cycles of Dynabeads. 相似文献
18.
Stephen Millam Alan T. H. Burns Trevor J. Hocking 《Plant Cell, Tissue and Organ Culture》1991,24(1):43-47
The effects of three different general purification protocols have been assessed quantitatively using mesophyll protoplasts of Brassica napus. Within the initial sample two distinct sub-populations were determined. The methods used influenced the ratio of the vacuolated to chloroplastic type protoplast sub-populations. Overall recovery rates of the initial sample varied according to the method used from 38% to 27%, but the relative recovery of the sub-populations varied considerably with a purified ratio of between 1.0:0.78 to 1.0:7.0. Size distribution profiles of the initial and purified populations are also presented. 相似文献
19.
Liquefaction of dry and freshPalmaria palmata by food grade enzyme preparations and a purified endo--1,4-D-xylanase was studied.The endo--1,4-D-xylanase (EC 3.2.1.8) was purified to homogeneity from a commercial food grade enzyme prepared fromAspergillus niger. It has a molecular weight of 22 500, a pI of 3.5, is inactive toward corn arabinoxylan,p-nitrophenyl--D-xylose, carboxymethyl cellulose but shows a weak activity toward microcrystalline cellulose. It hydrolyzes oat and dulse xylan equally well in seawater and deionized water essentially into xylose and xylobiose. It is stable between pH 5.5 to 9.0 and 0 to 30 °C and its activity is optimal at pH 4.5–5.5 and 40–60 °C. It has a Km of 2.2 and 2.8 mg ml-1 and Vmax of 3600 and 3900 nkat mg-1 of protein on oat and dulse xylan, respectively.Acetate buffer, deionized water and seawater alone extracted 62.6 to 64.5 % of the dry weight of dry dulse, but the use of commercial food grade enzyme preparations or the purified xylanase improved liquefaction to 81.2–87.1 %. Xylose and galactose were the only sugars present in the soluble extracts. Deionized and seawater extracted 58.8–52.7 and 39.1–42.2% of the dry weight of the fresh algae collected in fall and summer, respectively. Only galactose was found in the seawater extract, while some xylose with galactose were measured in the deionized water extract of the fresh autumn algal sample. Purified and crude xylanase improved liquefaction of fresh algae to 79.8–81.4 and 71.9–77.9% of the fresh dry weight (fall and summer, respectively) in deionized and seawater, respectively, and increased the xylose content of the soluble fractions. Polysaccharides in the soluble residues were composed of 1,3/1,4-linked xylose, 1-linked galactose (floridoside) and 1,4-linked glucose (cellulose) and contained essentially 1,4-linked xylose and 1,4-linked glucose in insoluble fractions obtained after enzymatic treatment.The use of xylanase-containing food grade enzyme preparations improves liquefaction ofPalmaria palmata, particularly from fresh alga. This study indicates that processing such as drying may modify markedly the solubility ofP. palmata cell wall polysaccharides, which would imply the existence of some organization and/or other components in the fresh cell wall that lower xylan solubility in seawater. 相似文献
20.
将去除信号肽的人肿瘤坏死因子-α(Tumor necrosis factor-α,TNF-α)cDNA插入到带有原核增强子样序列Px的新型表达载体pBV320中,使TNF cDNA 5′端直接置于大肠杆菌trp启动子下游,采用37℃恒温培养,使TNF在大肠杆菌中获得了高效表达,表达活性达1.35(±0.17)×10~6U/L菌液。表达的TNF-α对L929细胞的毒性作用可被抗人肿瘤坏死因子-α的单克隆抗体所中和。表达菌裂解液作SDS-聚丙烯酰胺凝胶电泳,显示有一条分子量与TNF分子量吻合、约为17000道尔顿的蛋白带。利用DEAE-Sepharose阴离子交换层析及Sephacryl S-200凝胶过滤对上述重组人TNF-α进行纯化,获得电泳纯产品,比活性为1.48×10~6U/mg。 相似文献