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排序方式: 共有92条查询结果,搜索用时 218 毫秒
1.
目的以重组人钠/碘转运体(hNIS)基因转染结肠癌SW480细胞并检测hNIS mRNA及蛋白的表达,为放射性碘治疗非甲状腺肿瘤提供新思路。方法将构建好的重组质粒(pcDNA3.1+-hNIS)进行酶切、测序鉴定并扩增、提取。SW480细胞分为重组质粒(pcDNA3.1+-hNIS)转染组、空白质粒(pcDNA3.1+)转染组、空白对照组,转染后以RT-PCR和Western blot检测各组细胞hNIS mRNA和蛋白的表达。结果酶切和测序结果显示插入的hNIS基因大小和方向均正确。RT-PCR和Western blot显示重组质粒转染组SW480细胞可见hNIS mRNA和蛋白的表达,而空白对照组和空白质粒转染组均未检测到hNIS mRNA和蛋白的表达。结论脂质体法可有效地将hNIS基因转染至SW480细胞并成功表达hNIS蛋白。 相似文献
2.
Direct Blue 71 staining as a destaining‐free alternative loading control method for Western blotting
Li Zeng Jing Guo Hong‐Bo Xu Rongzhong Huang Weihua Shao Liu Yang Mingju Wang Jianjun Chen Peng Xie 《Electrophoresis》2013,34(15):2234-2239
In Western blotting, a suitable loading control is indispensable for correcting errors in the total amount of loaded protein. Immunodetection of housekeeping proteins and total protein staining have traditionally been used as loading control methods. Direct Blue 71 (DB71) staining—a novel, sensitive, dye‐binding staining method compatible with immunodetection—may offer advantages over these traditional loading control methods. Three common neuroscientific samples (human plasma, human oligodendrocytes, and rat brain) were employed to assess DB71 staining as a loading control method for Western blotting. DB71, CBB, one traditional housekeeping protein, and one protein of interest were comparatively assessed for reliability and repeatability and linear dynamic range over 2.5–40 μg of protein loaded. DB71's effect on the reliability and repeatability and linear dynamic range of immunoreaction were also assessed. Across all three sample types, DB71 was either equivalent or superior to CBB and housekeeping protein‐based methods in terms of reliability and repeatability and linear dynamic range. Across all three sample types, DB71 staining did not impair the reliability and repeatability or linear dynamic range of immunoreaction. Our results demonstrate that the DB71 staining can be used as a destaining‐free alternative loading control method for Western blotting. 相似文献
3.
András Kovács Zoltán Patai András Guttman János Kádas László Takács István Kurucz 《Electrophoresis》2013,34(20-21):3064-3071
Immunization with complex mixtures, like the human plasma resulted in the generation of cloned mAb libraries (PlasmaScan? and QuantiPlasma? libraries, with >1000 individual mAbs) reacting with a nonredundant set of antigenic epitopes. mAb proteomics refers to quasi‐hypothesis‐free profiling of plasma samples with nascent or cloned mAb libraries for the discovery of disease‐specific biomarkers. Once mAbs with biomarker potential have been identified, the next task is the determination of cognate antigens recognized by the respective mAbs. To determine the cognate protein antigen corresponding to each individual mAbs in the cloned mAb libraries, we have separated human plasma by consecutive steps of desalting and various chromatography procedures. The process resulted in 783 fractions, which we termed “Analyte Library” (AL). The AL represents the human plasma proteome in relatively low‐protein complexity fractions. Here, to determine the utility of the AL, we selected ten plasma proteins and checked for their presence in the fractions. Among the ten cases, the distribution of four selected plasma proteins matched expectations, as these proteins were present only in a few fractions corresponding to their physical, chemical, and biochemical properties. However, in six cases, we observed “smear” ‐like distribution or complete absence of the proteins, suggesting that protein–protein interactions or protein variants may alter the observed plasma distribution profiles. Nevertheless, we conclude that the AL is an efficient, high throughput tool to complement the mAb biomarker discovery process with cognate protein antigen identification for each mAbs. 相似文献
4.
Zhen Wang Ziyang Li Tian Su Xiao Han Zhanwu Hou Yupeng Zheng Jiachen Liu Jun Xu Jeffy Yang Huadong Liu 《Electrophoresis》2021,42(6):793-799
Western blot (protein immunoblot) is a widely used analytical technique in molecular biology. Utilizing the specific recognizing primary antibody, proteins immobilized on various matrix are investigated by subsequent visualization steps, for example, by the horse radish peroxidase conjugated secondary antibody incubation. Methods to improve the sensitivity in protein identification or quantification are appreciated by biochemists. Herein, we report a new strategy to amplify Western blot signals by constructing a probe with proximal labeling and IgG targeting abilities. The R118G mutation attenuated the biotin-AMP binding affinity of the bacterial biotin ligase BirA*, offering a proximity-dependent labeling ability, which could be used as a signal amplifier. We built a BirA*-protein A fusion protein (BioEnhancer) that specifically binds to IgG and adds biotin tags to its proximal amine groups, enhancing the immunosignal of target proteins. In our experiments, the BioEnhancer system amplified the immunosignal by tenfold compared to the standard western blot. Additionally, our strategy could couple with other signal enhancement methods to further increase the western blot sensitivity. 相似文献
5.
Southwestern blotting is when a DNA sequence is used to probe DNA-binding proteins on an electrophoretic gel blot. It would be highly desirable to be able to probe a blot repeatedly with different DNA sequences. Alkaline phosphatase can remove 5'-phosphoryl groups from DNA and radiolabeled 5'-(32)P-DNA probes are commonly used in Southwestern blotting. Here is shown that once probed, the radioisotope signal on the blot can be effectively removed by brief digestion with alkaline phosphatase, and the blot can then be repeatedly probed at least six times with different DNA probes. This exceeds the repetitions possible with another commonly used method using SDS. The technique can be used with either one-dimensional or multi-dimensional Southwestern blots and does not have a large effect on the phosphorylation state of the blotted proteins. An alternative method using T4 polynucleotide kinase stripping is also introduced but was less well characterized. 相似文献
6.
7.
Hydatid cyst fluids (HCF) crude extracts from camels and sheep slaughtered in Riyadh region, KSA were subjected to Sodium Dodecyl Sulfate–Polyacrylamide Gel Electrophoresis (SDS–PAGE) and Western blot analysis. Sera from 17 confirmed human cases of hydatidosis, 25 patients with other parasitic infections and 10 clinically healthy subjects were used to evaluate the diagnostic value of the different antigenic fractions of these extracts. Immunoblotting results revealed that, at least 11 major discrete protein fractions (110–8 kDa) were recognized by sera from hydatidosis patients, sera from patients with other parasitic diseases showed cross-reactivity with few of these bands. The cluster of bands (38–35 kDa) that may be a breakdown of “Arc 5” antigen (39–38 kDa) was detected by 100% and 94% of sera from hydatidosis cases with HCF extracts from camel and sheep, respectively. This cluster showed also some cross reactivity (20% and 8%) with control sera from patients with other parasitic infections with camel and sheep HCF extracts, respectively. Polypeptides at 24–22, 16 and 8 kDa which may probably correspond to antigen B subunits were also identified by all samples from hydatidosis patients with sheep HCF extracts and by 100%, 65% and 74% with camel HCF extracts respectively. Sera from control subjects did not react with any of these polypeptides (24–22, 16 and 8 kDa). According to our results, the identified molecular weight bands (16 and 8 kDa using HCF crude extracts from sheep and 24–22 kDa using HCF crude extracts either from camel or sheep) represent good candidates for immunodiagnosis of hydatidosis. 相似文献
8.
陕南公路软弱变质岩边坡变形破坏特征的研究 总被引:1,自引:0,他引:1
陕西省是软弱变质岩分布广泛的省份之一。近年来,随着工程建设数量的增多和规模的加大,软弱变质岩斜坡灾害频繁发生,造成的损失也逐年增加。本文通过对陕南316国道早阳-蜀河段的实地调查,归纳了该路段软弱变质岩边坡的变形破坏特征,总结出顺层滑动、弯曲-倾倒、楔形体滑动、溃曲破坏以及滑移-拉裂5种典型的病害模式,并对每种变形破坏模式进行了具体的实例分析,从而为边坡成灾预警和选择经济有效的治理对策奠定基础。 相似文献
9.
伊犁河南岸滩地水文地质条件及其开发利用 总被引:2,自引:0,他引:2
董力民 《新疆大学学报(理工版)》1998,15(2):66-69
伊犁河南岸滩地面积广大,水土资源丰富,稍加投资改造即可利用,因此研究伊犁河滩地水土资源的开发利用,有其重要意义,本文以大量的第一手资料阐明了滩地的水文地质条件,分析和研究地下水的资源量,可开采量、排水问题及地下水资源的开发利用。 相似文献
10.
The allergenicity and structural changes of silver carp allergens influenced by high-pressure treatment were studied. We treated the allergens at 100, 200 and 300 MPa for 10, 30 and 60 min at 20° C, used SDS–PAGE to separate the proteins and recognized the allergens by western blotting. Circular dichroism analysis was performed to characterize the structural change. From our study, we can determine that high-pressure treatment did not change the subunit composition, molecular weight or the allergenicity of silver carp allergens, but it did change the structure of the allergens. 相似文献