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1.
While in the companion paper (Tonani, C. & Righetti, P. G., Electrophoresis 1991, 12, 1011-1021) we gave the general outline of our new computer program, immobilized pH gradients (IPG) simulator, able to simulate and optimize linear pH gradients for isoelectric focusing in immobilized pH gradients, in the present report we extend the application of such a program to: (i) convex exponential gradients, (ii) logarithmic and (iii) polynomial gradients. Such gradients are meant to give equal space to protein spots in complex protein mixtures (e.g., cell lysates, biological fluids) and follow the statistical distribution of protein pI values along the pH axis. They will prove of fundamental importance in two-dimensional maps, both because they optimize the spreading of spots in the two-dimensional plane and because of the excellent reproducibility of immobilized pH gradients. The following concave exponential recipes are given: pH 3-8, pH 3-9, pH 3-10, pH 3-11, pH 4-7, pH 4-8, pH 4-9, pH 4-10, pH 4-11, pH 5-8, pH 5-9, and pH 5-10, as well as the most extended pH 2.5-11 interval. Two interesting logarithmic gradients are described: pH 3-6 and pH 3-7 and one sigmoidal (derived with a polynomial of 5th degree): pH 3-11. 相似文献
2.
A.Corsico Coda G. Desimoni A.Gamba Invernizzi P.P. Righetti G. Tacconi 《Tetrahedron letters》1985,26(26):3137-3140
The difference between thermal, AlCl3- and CF3 COOH-catalyzed reaction of (E)-4-benzal-1-phenyl-5-pyrazolone and 2,3-dimethylbutadiene is discussed in terms of Diels-Alder heterodiene reactivity. 相似文献
3.
E Wenisch C Tauer A Jungbauer H Katinger M Faupel P G Righetti 《Journal of chromatography. A》1990,516(1):133-146
Isoforms of human monoclonal antibodies against the gp-41 of AIDS virus and of human recombinant superoxide dismutase have been purified to homogeneity by isoelectric focusing (IEF) in a multi-compartment electrolyser with isoelectric, immobiline membranes. This system allows the processing of large sample volumes and gram-scale protein loads and can resolve isoforms as close as 0.001 in pI difference. The purification progress was usually monitored by analytical IEF in immobilized pH gradients (IPG). Capillary zone electrophoresis (CZE) was applied to the monitoring of the content of each chamber of the electrolyser. CZE was found to be superior in terms of speed of analysis and quantification (but only by UV reading at 200-210 nm, i.e., in the region of the peptide bond) but, notwithstanding the millions of theoretical plates reported, was no match for the resolving power of IPGs, at least for protein analysis. When compared also with chromatofocusing, the resolving power decreases in the order IPG greater than CZE much greater than chromatofocusing. 相似文献
4.
The stereoisomers (1–4) of the tetrahydropyran[2′,3′:2,3] 2,3-dihydropyran[6,5-c] pyrazole system equilibrate in deuterated trifluoroacetic acid at 35°.The mechanism which causes incorporation of deuterium in the 4a-position is discussed.The isomers distribution is rationalized in terms of steric and electronic interactions, and the energy values for the interaction between the phenyl group in 5 and the substituent in position 6 on the pyrazole ring are given. 相似文献
5.
Oxidation of cysteine to cysteic acid in proteins by peroxyacids, as monitored by immobilized pH gradients. 总被引:1,自引:0,他引:1
It has often been debated whether the presence of persulfate in a polyacrylamide gel could lead to the oxidation of cysteine (Cys) in proteins to cysteic acid. In fact, direct incubation of bovine serum albumin (BSA) with peroxodisulfate and periodate barely alters the isoelectric point (pI) and does not produce any cysteic acid. In contrast, caroate (peroxomonosulfate) and perphthalate strongly lower the pI of BSA. In the former case it as demonstrated that 4-Cys (of a total of 35) were converted into cysteic acid. Perphthalate was found to be, by far, the strongest oxidant: 15 (of 35) Cys residues were oxidized to cysteic acid and all methionine groups were destroyed. 相似文献
6.
A novel method is reported for screening for point mutations in genomic DNA: free-zone capillary electrophoresis in very acidic buffers. This method exploits the charge difference among the four different bases (C, T, A, G) in a pH window between 2.5 and 3.5, where the four titration curves fan out. The method is applied to the detection of the beta-39 missense mutation in the beta-globin gene in thalassemias. A 60-mer fragment straddling the mutation site has been amplified. In an isoelectric buffer (iminodiacetic acid) of pH 3.3, partial resolution between the wild type and mutated strands is obtained. In a pH 3.0 phosphate buffer, baseline resolution is achieved between the two strands in a heterozygous individual. Due to the short size of the amplified fragment, this method can only be applied to routine screening for known mutations because resolution was lost in a fragment 100 bases long. 相似文献
7.
The zone spreading caused by a transverse pH profile due to a temperature gradient through the thickness of a gel slab is estimated. The temperature difference (delta T) between the upper and lower gel surfaces can be calculated as a function of the electric power dissipated in the gel and the gel dimensions. It is found that when delta T is only 1 degree C the zone spreading due to this thermal excursion is as high as 0.5 mm. Thus, an admissible delta T is found to be equal to 0.2 degrees C, since this corresponds to a thermal zone spreading of only 0.1 mm, i.e. the same order of magnitude as the spatial resolution of a laser scanner. A thermal zone spreading of 0.1 mm is compatible with a resolving power of 0.01 pH unit, the current limit of conventional isoelectric focusing in amphoteric buffers. A requirement for the thickness of a gel slab is formulated: e.g., at 40 W applied power (over a gel surface area of 25 x 11 cm), a thermal zone spreading limited to 0.1 mm can only be obtained with a gel thickness of approximately 170 microns. 相似文献
8.
Morelos A Albuquerque IF Bondar NF Carrigan RA Chen D Cooper PS Lisheng D Denisov AS Dobrovolsky AV Dubbs T Endler AM Escobar CO Foucher M Golovtsov VL Gottschalk H Gouffon P Grachev VT Khanzadeev AV Kubantsev MA Kuropatkin NP Lach J Lang Pengfei Li Chengze Li Yunshan Luksys M Mahon JR McCliment E Newsom C Pommot Maia MC Samsonov VM Schegelsky VA Shi Huanzhang Smith VJ Tang Fukun Terentyev NK Timm S Tkatch II Uvarov LN Vorobyov AA Yan Jie Zhao Wenheng Shuchen Z Zhong Yuanyuan 《Physical review letters》1993,71(21):3417-3420
9.
Morelos A Albuquerque IF Bondar NF Carrigan RA Chen D Cooper PS Dai Lisheng Denisov AS Dobrovolsky AV Dubbs T Endler AM Escobar CO Foucher M Golovtsov VL Gottschalk H Gouffon P Grachev VT Khanzadeev AV Kubantsev MA Kuropatkin NP Lach J Lang Pengfei Li Chengze Li Yunshan Luksys M Mahon JR McCliment E Newsom C Pommot Maia MC Samsonov VM Schegelsky VA Shi Huanzhang Smith VJ Tang Fukun Terentyev NK Timm S Tkatch II Uvarov LN Vorobyov AA Yan Jie Zhao Wenheng Zheng Shuchen Zhong Yuanyuan 《Physical review letters》1993,71(14):2172-2175
10.
A new method is described for producing highly porous polyacrylamide matrices: polymerization in presence of a preformed hydrophilic polymer. If a standard mixture of monomers (e.g., 5%T, 4%C) is polymerized in presence of, e.g., polyethylene glycol (PEG) 10 kDa, lateral chain aggregation occurs, with formation of large pore sizes. In PEG 10 kDa, the transition from a small- to a large-pore gel is clearly apparent at 0.5% PEG addition and reaches a plateau already at 2.5% PEG. Even with shorter PEG fragments (6.2 and 1 kDa) this transition occurs, but with progressively larger amounts of PEG in solution (up to 25% for the 1 kDa species). Other polymers such as hydroxymethyl cellulose (1000 kDa) and polyvinyl-pyrrolidone (360 kDa and 25 kDa) are also able to elicit this phenomenon. It appears that lateral chain aggregation (before the cross-linking event) is induced via intra-chain hydrogen bonding, since urea and temperature strongly inhibit it, whereas tetramethylurea (an agent quenching hydrophobic interactions) does not hamper it. By scanning electron microscope, it is found that the maximum pore size obtained in a 5%T, 4%C gel in presence of 2.5% PEG 10 kDA is of the order of 0.5 micron, whereas the same 5%T, 4%C control gel would have an average pore diameter of 5 nm. Thus, an increment of pore size of about 2 orders of magnitude is obtained: in these new matrices, a 21000 bp DNA fragment exhibits a much greater migration than in a control gel in which the sample is entrapped at the application site. 相似文献