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51.
Igor Chernukhin Sung Yun Kang Sam Brown Svetlana Gretton Claudia Fabiola Mendez-Catala Dave Cowieson Elena Klenova 《Analytical biochemistry》2011,(2):183
Chromatin immunoprecipitation (ChIP) is an important technique in the study of DNA/protein interactions. The ChIP procedure, however, has limitations in that it is lengthy, can be inconsistent, and is prone to nonspecific binding of DNA and proteins to the bead-based solid-phase matrices that are often used for the immunoprecipitation step. In this investigation, we examined the utility of a new matrix for ChIP assays, BioVyon Protein A, a solid support based on porous polyethylene. In ChIP experiments carried out using two antibodies and seven DNA loci, the performance of BioVyon Protein A was significantly better, with a greater percentage of DNA pull-down in all of the assays tested compared with bead-based matrices, Protein A Sepharose, and Dynabeads Protein A. Furthermore, the rigid porous disc format within a column made the BioVyon matrix much easier to use with fewer steps and less equipment requirements, resulting in a significant reduction in the time taken to process the ChIP samples. In summary, BioVyon Protein A provides a column-based assay method for ChIP and other immunoprecipitation-based procedures; the rigid porous structure of BioVyon enables a fast and robust protocol with higher ChIP enrichment ratios. 相似文献
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Irene EM Bultink D?rte Hamann Marc A Seelen Margreet H Hart Ben AC Dijkmans Mohamed R Daha Alexandre E Voskuyl 《Arthritis research & therapy》2007,8(6):R183
Infection imposes a serious burden on patients with systemic lupus erythematosus (SLE). The increased infection rate in SLE
patients has been attributed in part to defects of immune defence. Recently, the lectin pathway of complement activation has
also been suggested to play a role in the occurrence of infections in SLE. In previous studies, SLE patients homozygous for
mannose-binding lectin (MBL) variant alleles were at an increased risk of acquiring serious infections in comparison with
patients who were heterozygous or homozygous for the normal allele. This association suggests a correlation between functional
MBL level and occurrence of infections in SLE patients. We therefore investigated the biological activity of MBL and its relationship
with the occurrence of infections in patients with SLE. Demographic and clinical data were collected in 103 patients with
SLE. Functional MBL serum levels and MBL-induced C4 deposition were measured by enzyme-linked immunosorbent assay using mannan
as coat and an MBL- or C4b-specific monoclonal antibody. The complete MBL-dependent pathway activity was determined by using
an assay that measures the complete MBL pathway activity in serum, starting with binding of MBL to mannan, and was detected
with a specific monoclonal antibody against C5b-9. Charts were systematically reviewed to obtain information on documented
infections since diagnosis of SLE. Major infections were defined as infections requiring hospital admission and intravenous
administration of antibiotics. In total, 115 infections since diagnosis of lupus, including 42 major infections, were documented
in the 103 SLE patients (mean age 41 ± 13 years, mean disease duration 7 ± 4 years). The percentage of SLE patients with severe
MBL deficiency was similar to that in 100 healthy controls: 13% versus 14%, respectively. Although deposition of C4 to mannan
and MBL pathway activity were reduced in 21% and 43% of 103 SLE patients, respectively, neither functional MBL serum levels
nor MBL pathway activity was associated with infections or major infections in regression analyses. In conclusion, SLE patients
frequently suffer from infections, but deficiency of functional MBL does not confer additional risk. 相似文献
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Immobilization of native proteins, retaining their activity, on the solid support is often crucial for a variety of biochemical assays involving protein-protein interactions. In this study we describe a technique which allows binding of both complex (protein kinase CK2) and simple (calf intestine alkaline phosphatase, CIP) enzymes to the solid support without denaturization of the proteins. This method is based on the covalent cross-linking of the enzymes to the bifunctional resin, containing the secondary amino and thiol groups, in a coupling reaction with the imidoester dimethyl pimelimidate hydrochloride. Both enzymes in their bound form were active in the specific biochemical assays. We also found that the CK2 and CIP resins did not change their activity for at least 3 months, and the quality of these resins were not affected by high salts or reducing agents. Thus, this method can be recommended for general use to generate active enzymes coupled to the solid support. 相似文献
59.
Endogenous chloride channels of insect sf9 cells. Evidence for coordinated activity of small elementary channel units 总被引:1,自引:0,他引:1 下载免费PDF全文
EH Larsen SE Gabriei MJ Stutts J Fullton EM Price RC Boucher 《The Journal of general physiology》1996,107(6):695-714
The endogenous Cl- conductance of Spodoptera frugiperda (Sf9) cells was studied 20-35 h after plating out of either uninfected cells or cells infected by a baculovirus vector carrying the cloned beta-galactosidase gene (beta-Gal cells). With the cation Tris+ in the pipette and Na+ in the bath, the reversal potential of whole-cell currents was governed by the prevailing Cl- equilibrium potential and could be fitted by the Goldman-Hodgkin-Katz equation with similar permeabilities for uninfected and beta-Gal cells. In the frequency range 0.12 < f < 300 Hz, the power density spectrum of whole-cell Cl- currents could be fitted by three Lorentzians. Independent of membrane potential, >50% of the total variance of whole-cell current fluctuations was accounted for by the low frequency Lorentzian (fc = 0.40 +/- 0.03 Hz, n = 6). Single-Cl- channels showed complex gating kinetics with long lasting (seconds) openings interrupted by similar long closures. In the open state, channels exhibited fast burst-like closures. Since the patches normally contained more than a single channel, it was not possible to measure open and closed dwell-time distributions for comparing single-Cl- channel activity with the kinetic features of whole-cell currents. However, the power density spectrum of Cl- currents of cell-attached and excised outside-out patches contained both high and low frequency Lorentzian components, with the corner frequency of the slow component (fc = 0.40 +/- 0.02 Hz, n = 4) similar to that of whole-cell current fluctuations. Chloride channels exhibited multiple conductance states with similar Goldman-Hodgkin-Katz-type rectification. Single-channel permeabilities covered the range from approximately 0.6.10(-14) cm5/s to approximately 6.10(-14) cm3/s, corresponding to a limiting conductance (gamma 150/150) of approximately 3.5 pS and approximately 35 pS, respectively. All states reversed near the same membrane potential, and they exhibited similar halide ion selectivity, P1 > PCl approximately PBr. Accordingly, Cl- current amplitudes larger than current flow through the smallest channel unit resolved seem to result from simultaneous open/shut events of two or more channel units. 相似文献
60.
YK Onno Teng Gillian Wheater Vanessa E Hogan Philip Stocks EW Nivine Levarht Tom WJ Huizinga Rene EM Toes Jacob M van Laar 《Arthritis research & therapy》2012,14(2):R57