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201.
人心肌肌钙蛋白I-C融合蛋白在大肠杆菌中的可溶性表达及纯化 总被引:1,自引:0,他引:1
提取人心肌组织总RNA,RT-PCR扩增出人心肌肌钙蛋白I(cardiac troponin I,cTnI)和肌钙蛋白C(TnC)基因,利用人工合成的可编码19个中性氨基酸残基为主的DNA序列(Linker),将cTnI和TnC基因连接,插入到质粒pET15b中,将鉴定正确的重组pET15b质粒转化入大肠杆菌BL21(DE3)pLysS中,用异丙基-β-D–硫代半乳糖苷(IPTG)诱导蛋白质表达.结果在大肠杆菌中成功实现了全长cTnI-C融合蛋白(cTnI-linker-TnC)的稳定可溶性高表达,在摇瓶中表达量可达21 mg/L.利用目的蛋白N端的组氨酸“标签”(His-tag),经一步Ni2+-Sepharose柱亲和层析纯化,得到了聚丙烯酰胺凝胶电泳(PAGE)纯蛋白质.经初步研究,cTnI-C具有较好的免疫原性和稳定性,有望成为cTnI测定系统可溯源的候选参考物质,推进cTnI检测的标准化进程. 相似文献
202.
203.
链霉菌SO1菌株几丁质酶的纯化及性质 总被引:3,自引:0,他引:3
由链霉菌(Streptomycessp.)SO1菌株产生的几丁质酶(Citinase)经硫酸铵盐析、OEAE纤维素柱层析、SephadexG-100柱层桥分离纯化后,得到SDS-PAGE均一样品。用SDS-PAG测得纯化后的几丁质酶分子量为41Ku,用PAGEIEF测得等电点PI为5.4。酶反应的最适pH值为6.0,最适温度为50℃,在pH5.0~9.0、温度30~50℃时酶活性比较稳定。在相当于0.1mol/LNaCl的离子强度下酶活性最高。金属离子中的Mg2+、Ca 相似文献
204.
205.
植物生物反应器是一种新兴的重组蛋白表达系统,是分子农业的核心内容之一。本研究在本氏烟草(Nicotiana benthamiana)中表达了抗八肽(DYKDDDDK, FLAG)标签抗体,并对其进行纯化与鉴定。通过多次免疫小鼠获得高效价抗FLAG抗体并测出其编码序列,然后亚克隆至植物DNA病毒表达载体,最后通过农杆菌介导转染烟草叶片。经Western blotting检测了转染后2−9 d抗体的表达情况:3 d后FLAG抗体开始在烟草叶片中表达,5 d后表达量达到峰值,每千克鲜叶估计可表达66 mg FLAG抗体。抗体经过分离纯化后浓缩为1 mg/mL,按1:10 000稀释仍可识别1 ng/mL的抗原,表明植物生产的FLAG抗体具有高亲和力。植物生物反应器可用于生产高亲和力抗体,并具有简易、成本低和生产周期短等特点,具有很高的应用价值。 相似文献
206.
Célia Parinot Quentin Rieu Jonathan Chatagnon Silvia C. Finnemann Emeline F. Nandrot 《Journal of visualized experiments : JoVE》2014,(94)
Analysis of one of the vital functions of retinal pigment epithelial (RPE) cells, the phagocytosis of spent aged distal fragments of photoreceptor outer segments (POS) can be performed in vitro. Photoreceptor outer segments with stacks of membranous discs containing the phototransduction machinery are continuously renewed in the retina. Spent POS are eliminated daily by RPE cells. Rodent, porcine/bovine and human RPE cells recognize POS from various species in a similar manner. To facilitate performing large series of experiments with little variability, a large stock of POS can be isolated from porcine eyes and stored frozen in aliquots. This protocol takes advantage of the characteristic of photopigments that display an orange color when kept in the dark. Under dim red light, retinae are collected in a buffer from opened eyecups cut in halves. The retinal cell suspension is homogenized, filtered and loaded onto a continuous sucrose gradient. After centrifugation, POS are located in a discrete band in the upper part of the gradient that has a characteristic orange color. POS are then collected, spun, resuspended sequentially in wash buffers, counted and aliquoted. POS obtained this way can be used for phagocytosis assays and analysis of protein activation, localization or interaction at various times after POS challenge. Alternatively, POS can be labeled with fluorophores, e.g., FITC, before aliquoting for subsequent fluorescence quantification of POS binding or engulfment. Other possible applications include the use of modified POS or POS challenge combined with stress conditions to study the effect of oxidative stress or aging on RPE cells. 相似文献
207.
《Bioscience, biotechnology, and biochemistry》2013,77(11):1890-1891
The effects of the ovarian hormones progesterone and estrone on the conversion of tryptophan to nicotinamide in rats were investigated. Female rats were fed for 35 days with a 20% casein diet, or with a 20% casein diet containing 0.1% progesterone, or 0.001% estrone, or 0.1% progestrone and 0.001% estrone. The conversion ratio of tryptophan to nicotinamide on the last day of the experiment was 2% in the groups fed with the 20% casein diet and the diet containing 0.1% progesterone, but around 1.2% in the group fed with 0.001% estrone, and 0.7% in the group fed with 0.1% progesterone and 0.001% estrone. These results demonstrated that administration of ovarian hormones significantly decreased the conversion of tryptophan to nicotinamide. 相似文献
208.
We describe the complete process of AcroPrep Advance Filter Plates for 96 plasmid preparations, starting from prokaryotic culture and ending with high purity DNA. Based on multi-well filtration for bacterial lysate clearance and DNA purification, this method creates a streamlined process for plasmid preparation. Filter plates containing silica-based media can easily be processed by vacuum filtration or centrifuge to yield appreciable quantities of plasmid DNA. Quantitative analyses determine the purified plasmid DNA is consistently of high quality with average OD260/280 ratios of 1.97. Overall, plasmid yields offer more pure DNA for downstream applications, such as sequencing and cloning. This streamlined method of using AcroPrep Advance Filter Plates allows for manual, semi-automated or fully-automated processing. 相似文献
209.
A trypsin and chymotrypsin inhibitor was partially purified from Bauhenia purpurea seeds and separated from a second inhibitor by Ecteola cellulose chromatography. The factor inhibited bovine trypsin and chymotrypsin as well as pronase trypsin and elastase. It formed a complex with trypsin and with chymotrypsin, but a ternary complex could not be detected. Differences were detected in the effect on trypsin and on chymotrypsin, although one enzyme interfered with the inhibition of the other. The results obtained point to two active centers on the inhibitor for the trypsin and chymotrypsin inhibition such that the one cannot complex with the inhibitor after this inhibitor had complexed with the other. 相似文献
210.
Alkaline phosphatase isozymes in the midgut of silkworm: purification of high pH-stable microvillus and labile cytosolic enzymes 总被引:1,自引:0,他引:1
Nahoko Okada Masaaki Azuma Masaharu Eguchi 《Journal of comparative physiology. B, Biochemical, systemic, and environmental physiology》1989,159(2):123-130
Summary Genetically defined alkaline phosphatase (ALP) isozymes from the larval midgut tissues ofBombyx mori were purified and characterized. The membrane-bound form (m-ALP) was solubilized with 1% Triton X-100, then purified by DEAE-Sephacel, Con A-Sepharose 4B and Ultrogel AcA 34 column chromatography. The soluble form (s-ALP) was purified by DEAE-Sephacel, epoxy Toyopearl coupled with phosphonic acid and Ultrogel AcA 34 column chromatography. About 840- and 650-fold purification were achieved for m-ALP and s-ALP, respectively, and both ALPs were homogeneous as judged by poly-acrylamide gel electrophoresis. Both forms were found to be similar (MW=68 000 in gel permeation chromatography, and a single subunit as a monomer in denaturing SDS-polyacrylamide gels with MW=58 000 for m-ALP and MW=61 000 for s-ALP). The pH optima of ALPs were shown to lie at 10.9 (m-ALP) and 9.8 (s-ALP), the former being extremely stable even in pH 10–12 which accords with the physiological milieu inBombyx midgut lumen. The Km values of the m-ALP and s-ALP forp-nitrophenyl phosphate were 1.99 and 1.49 mM, respectively. Both ALPs had a similar substrate specificity.l-Cysteine strongly inhibited both ALPs, but inhibitory effects ofl-phenylalanine,l-homoarginine andl-leucine were undetectable for s-ALP and very weak for m-ALP. A comparison of enzymatic properties on two ALPs suggested that each isozyme plays different roles.Abbreviations
m-ALP
membrane-bound alkaline phosphatase
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s-ALP
soluble alkaline phosphatase 相似文献