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111.
Komeiji Yuto; Uebayasi Masami; Someya Jun-ichiro; Yamato Ichiro 《Protein engineering, design & selection : PEDS》1992,5(8):759-767
The Ser88Cys mutant of the trp-repressor showed a lower affinityfor the corepressor than the wild-type repressor [G = 1.7 ±0.3 kcal/mol, Chou and Matthews (1989) J. Biol. Chem., 264,1831418319].A molecular dynamics/free energy cycle perturbation study wasperformed to understand the origin of the decreased affinity.A value (G = 1.58 ± 0.28 kcal/mol) comparable with theexperimental value was obtained by the simulation. Free energycomponent analysis revealed that destabilization of the vander Waals interaction between Ser88 and Trp109 (corepressor)mainly contributed to the decreased affinity of the mutant.The rotational transition of the hydroxyl (sulfhydryl) groupof Ser88 (Cys88) during the simulations affected the contributionsof Arg84 and water to the free energy change in the aporepressorand those of Arg84 and Trp 109 to that in the holorepressor.However, the contributions from different residues compensatedeach other, and the total free energy changes were almost invariablein the various simulations. 相似文献
112.
Bottomley Stephen P.; Popplewell Andrew G.; Scawen Michael; Wan Tommy; Sutton Brian J.; Gore Michael G. 《Protein engineering, design & selection : PEDS》1994,7(12):1463-1470
The stability and unfolding of an immunoglobulin (Ig) G bindingprotein based upon the B domain of protein A (SpAB) from Staphylococcusaureus were studied by substituting tryptophan residues at strategiclocations within each of the three a-helical regions (al-a3)of the domain. The role of the C-terminal helix, a3, was investigatedby generating two protein constructs, one corresponding to thecomplete SpAB, the other lacking a part of ct3; the Trp substitutionswere made in both one-and two-domain versions of each of theseconstructs. The fluorescence properties of each of the single-tryptophanmutants were studied in the native state and as a function ofguanidine-HCl-mediated unfolding, and their IgG binding activitieswere determined by a competitive enzyme-linked immunosorbentassay. The free energies of folding and of binding to IgG foreach mutant were compared with those for the native domains.The effect of each substitution upon the overall structure andupon the IgG binding interface was modelled by molecular graphicsand energy minimization. These studies indicate that (i) 3 contributesto the overall stability of the domain and to the formationof the IgG binding site in l and 2, and (ii) al unfolds first,followed by 2 and 3 together. 相似文献
113.
Ribosome display of mammalian receptor domains 总被引:2,自引:0,他引:2
Many mammalian receptor domains, among them a large number of potential therapeutic target proteins, are highly aggregation-prone upon heterologous expression in bacteria. This severely limits functional studies of such receptor domains and also their engineering towards improved properties. One of these proteins is the Nogoreceptor, which plays a central role in mediating the inhibition of axon growth and functional recovery after injury of the adult mammalian central nervous system. We show here that the ligand binding domain of the Nogoreceptor folds to an active conformation in ternary ribosomal complexes, as formed in ribosome display. In these complexes the receptor is still connected, via a C-terminal tether, to the peptidyl tRNA in the ribosome and the mRNA also stays connected. The ribosome prevents aggregation of the protein, which aggregates as soon as the release from the ribosome is triggered. In contrast, no active receptor was observed in phage display, where aggregation appears to prevent incorporation of the protein into the phage coat. This strategy sets the stage for rapidly studying defined mutations of such aggregation-prone receptors in vitro and to improve their properties by in vitro evolution using the ribosome display technology. 相似文献
114.
Recently some heat-shock proteins have been linked to functionsof chaperoning protein folding in vivo. Here currentexperimental evidence is reviewed and possible requirementsfor such an activity are discussed. It is proposed that onemode of chaperone action is to actively unfold misfolded orbadly aggregated proteins to a conformation from whkh they couldrefold spontaneously; that improperly folded proteins are recognizedby excessive stretches of solvent-exposed backbone, rather thanby exposed hydrophobic patches; and that the molecular mechanismfor unfolding is either repeated binding and dissociation (plucking)or translocation of the protein backbone through a binding cleft(threading), allowing the threaded chain to refoldspontaneously. The observed hydrolysis of ATP would providethe energy for active unfolding. These hypotheses can be appliedto both monomeric folding and oligomeric assembly and are sufficientlydetailed to be open to directed experimental verification. 相似文献
115.
Greenwood M.Jeffrey; Ong Edgar; Gilkes R.Neil; Warren R.Antony J.; Miller C.Robert Jr; Kilburn G.Douglas 《Protein engineering, design & selection : PEDS》1992,5(4):361-365
The endoglucanase CenA and the exoglucanase Cex from Cellulomonasfimi each contain a discrete cellulose-binding domain (CBD),at the amino-terminus or carboxyl-terminus respectively. Thegene fragment encoding the CBD can be fused to the gene of aprotein of interest. Using this approach hybrid proteins canbe engineered which bind reversibly to cellulose and exhibitthe biological activity of the protein partner. Alkaline phosphatase(PhoA) from Escherichia coli, and a ß-glucosidase(Abg) from an Agrobacterium sp. are dimeric proteins. The fusionpolypeptides CenA-PhoA and Abg-CBCcex are sensitive to proteolysisat the junctions between the fusion partners. Proteolysis resultsin a mixture of homo- and heterodimers; these bind to celluloseif one or both of the monomers carry a CBD, e.g. CenA-PhoA/CenA-PhoAand CenA-PhoA/PhoA. CBD fusion polypeptides could be used inthis way to purify polypeptides which associate with the fusionpartner. 相似文献
116.
Characterization of yam bean (Pachyrhizus spp.) Seeds as potential sources of high palmitic acid oil
W. J. Grüneberg F. D. Goffman L. Velasco 《Journal of the American Oil Chemists' Society》1999,76(11):1309-1312
Seeds from 22 accessions of the yam bean species Pachyrhizus ahipa (14 accessions), P. erosus (5), and P. tuberosus (3) were investigated for oil and protein contents, fatty acid composition of the seed oil, and the total tocopherol content
and composition. Plants from the accessions were grown under greenhouse conditions during one (P. erosus and P. tuberosus) or two years (P. ahipa). The pattern of the investigated seed quality traits was very similar in the three species. Yam bean seeds were characterized
by high oil (from about 20 to 28% in one environment) and protein contents (from about 23 to 34%). Seed oil contained high
concentrations of palmitic (from about 25 to 30% of the total fatty acids), oleic (21 to 29%), and linoleic acids (35 to 40%).
Levels of linolenic acid were very low, from about 1.0 to 2.5%. Total tocopherol content was relatively low in P. erosus (from 249 to 585 mg kg−1 oil) and P. tuberosus (from 260 to 312 mg kg−1 oil) compared with the levels found in P. ahipa grown under identical conditions (508 to 858 mg kg−1 oil). In all the samples, γ-tocopherol was predominant, accounting for more than 90% of the total tocopherol content. The
combination of high oil and protein contents, together with high palmitic acid, low linolenic acid, and high γ-tocopherol
concentration, makes these crops an interesting alternative as sources of high palmitic acid oil for the food industry. 相似文献
117.
Local polarity analysis: a sensitive method that discriminates between native proteins and incorrectly folded models 总被引:1,自引:0,他引:1
Luthardt Gudrun; Frommel Cornelius 《Protein engineering, design & selection : PEDS》1994,7(5):627-631
The evaluation of calculated protein structures is an importantstep in the protein design cycle. Known criteria for this assessmentof proteins are the polar and apolar, accessible and buriedsurface area, electrostatic interactions and other interactionsbetween the protein atoms (e.g. HO, S-S),atomic packing, analysisof amino acid environment and surface charge distribution. Weshow that a powerful test of accuracy of protein structure canbe derived by analysing the water contact of atoms and additionallytaking into account their polarity. On the basis of estimatedreference values of the polar fraction of typical globular proteinswith known structure (mean, SD and distribution), the evaluationof misfolded structures can be improved significantly. The referencevalues are derived by moving windows of different length (399amino acid residues) over the amino acid sequence. Model proteins,which are included in the Brookhaven protein structure databank,deliberately misfolded proteins, hypothetical proteins and predictedprotein structures are diagnosed as at least partially incorrectlyfolded. The local fault, mostly observed, is that polar groupsare buried too frequently in the interior of the protein. Thedatabase-derived quantities are useful in screening the designedproteins prior to experimentation and may also be useful inthe assessment of errors in the experimentally determined proteinstructures. 相似文献
118.
采用超声波—微波协同提取法提取芝麻渣中蛋白质,并用超滤法进行纯化.实验考察了影响粗蛋白提取率的固液比、溶液pH值、微波功率、提取时间等因素,确定了最佳提取条件;同时考察了超滤膜的截留分子质量以及压力对超滤效果的影响.结果表明:超声波—微波协同提取最佳条件为超声波功率40 W,固液比1∶20,溶液pH11,微波功率250 W、提取时间90 s,提取率约55.32%;采用10万截留分子质量的超滤膜在0.18 MPa压力下对芝麻渣蛋白质纯化后,蛋白质纯度提高了15.67%. 相似文献
119.
120.
Citrate-stabilized CdSe/CdS quantum dots as fluorescence probe for protein determination 总被引:1,自引:1,他引:0
A rapid, ultrasensitive and convenient fluorescence measurement technology based on the enhancement of the fluorescence intensity
resulting from the interaction of functionalized CdSe/CdS quantum dots (QDs) with bovine serum albumin (BSA) was proposed.
The citrate-stabilized CdSe/CdS (QDs) were synthesized by using Se powder and Na2S as precursors instead of any pyrophoric organometallic precursors. The modified CdSe/CdS QDs are brighter and more stable
against photobleaching in comparison with organic fluorophores. At pH 7.0, the fluorescence signal of CdSe/CdS is enhanced
by increasing the concentration of BSA in the range of 0.1–10 μg/mL, and the low detection limit is 0.06 μg/mL. A linear relationship
between the enhanced fluorescence peak intensity (ΔF) and BSA concentration (c) is established using equation ΔF=50.7c+16.4 (R=0.996 36). Results of determination for BSA in three synthetic samples are identical with the true values, and the recovery
(98.9%–102.4%) and relative standard deviation (RSD, 1.8%–2.5%) are satisfactory. 相似文献