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生物化学

Measurement of FNR-NrdI Interaction by Microscale Thermophoresis (MST) 通过微量热泳法(MST)测定FNR-NrdI的相互作用 作者:Ingvild Gudim, Marie Lofstad, Marta Hammerstad and Hans-Petter Hersleth日期:04/20/2017,浏览量:8938,Q&A: 0
This protocol describes how to measure protein-protein interactions by microscale thermophoresis (MST) using the MonolithTM NT.115 instrument (NanoTemper). We have used the protocol to determine the binding affinities between three different flavodoxin reductases (FNRs) and a flavodoxin-like protein, NrdI, from Bacillus cereus ...
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Determination of Hydrodynamic Radius of Proteins by Size Exclusion Chromatography 分子排阻色谱法测定蛋白质的流体动力学半径 作者:Valentina La Verde, Paola Dominici and Alessandra Astegno日期:04/20/2017,浏览量:27169,Q&A: 0
Size exclusion chromatography (SEC) or gel filtration is a hydrodynamic technique that separates molecules in solution as a function of their size and shape. In the case of proteins, the hydrodynamic value that can be experimentally derived is the Stokes radius (Rs), which is the radius of a sphere with the same hydrodynamic properties ( ...
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Expression, Purification and Crystallisation of the Adenosine A2A Receptor Bound to an Engineered Mini G Protein 与改造的迷你G蛋白结合的腺苷A2A受体的表达、纯化和结晶 作者:Byron Carpenter and Christopher G. Tate日期:04/20/2017,浏览量:8437,Q&A: 0
G protein-coupled receptors (GPCRs) promote cytoplasmic signalling by activating heterotrimeric G proteins in response to extracellular stimuli such as light, hormones and nucleosides. Structure determination of GPCR–G protein complexes is central to understanding the precise mechanism of signal transduction. However, these complexes are ...
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Expression and Purification of Mini G Proteins from Escherichia coli 迷你G蛋白在大肠埃希杆菌的表达和纯化 作者:Byron Carpenter and Christopher G. Tate日期:04/20/2017,浏览量:9447,Q&A: 0
Heterotrimeric G proteins modulate intracellular signalling by transducing information from cell surface G protein-coupled receptors (GPCRs) to cytoplasmic effector proteins. Structural and functional characterisation of GPCR–G protein complexes is important to fully decipher the mechanism of signal transduction. However, native G proteins are ...
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Affinity Purification of the RNA Degradation Complex, the Exosome, from HEK-293 Cells 来自HEK-293细胞的外泌体RNA降解复合物的亲和纯化 作者:Michal Domanski and John LaCava日期:04/20/2017,浏览量:6555,Q&A: 0
The RNA exosome complex plays a central role in RNA processing and regulated turnover. Present both in cytoplasm and nucleus, the exosome functions through associations with ribonucleases and various adapter proteins (reviewed in [Kilchert et al., 2016]). The following protocol describes an approach to purify RNA exosome complexes from ...
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癌症生物学

Melanoma Stem Cell Sphere Formation Assay 黑色素瘤干细胞球体形成测定 作者:Alessandra Tuccitto, Valeria Beretta, Francesca Rini, Chiara Castelli and Michela Perego日期:04/20/2017,浏览量:11163,Q&A: 0
Self-renewal is the ability of cells to replicate themselves at every cell cycle. Throughout self-renewal in normal tissue homeostasis, stem cell number is maintained constant throughout life. Cancer stem cells (CSCs) share this ability with normal tissue stem cells and the sphere formation assay (SFA) is the gold standard assay to assess stem ...
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Virtual Screening of Transmembrane Serine Protease Inhibitors 跨膜丝氨酸蛋白酶抑制剂的虚拟筛选 作者:Antti Poso, Topi Tervonen and Juha Klefström日期:04/20/2017,浏览量:7000,Q&A: 0
The human family of type II transmembrane serine proteases includes 17 members. The defining features of these proteases are an N-terminal transmembrane domain and a C-terminal serine protease of the chymotrypsin (S1) fold, separated from each other by a variable stem region. Recently accumulated evidence suggests a critical role for these ...
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细胞生物学

Time-lapse Observation of Chromosomes, Cytoskeletons and Cell Organelles during Male Meiotic Divisions in Drosophila 果蝇雄性减数分裂期间染色体、细胞骨架和细胞器的延时观察 作者:Karin Tanabe, Ryotaro Okazaki, Kana Kaizuka and Yoshihiro H. Inoue日期:04/20/2017,浏览量:6872,Q&A: 0
In this protocol, we provide an experimental procedure that perform time-lapse observation of intra-cellular structures such as chromosomes, cytoskeletons and cell organelles during meiotic cell divisions in Drosophila males. As primary spermatocyte is the largest dividing diploid cell in Drosophila, which is equivalent in size ...
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RNase Sensitivity Screening for Nuclear Bodies with RNA Scaffolds in Mammalian Cells 哺乳动物细胞中含RNA支架的核体的核糖核酸酶敏感性筛选 作者:Taro Mannen and Tetsuro Hirose日期:04/20/2017,浏览量:7732,Q&A: 0
The mammalian cell nucleus is highly organized and contains membraneless nuclear bodies (NBs) characterized by distinct resident factors. The NBs are thought to serve as sites for biogenesis and storage of certain RNA and protein factors as well as assembly of ribonucleoprotein complexes. Some NBs are formed with architectural RNAs (arcRNAs) as ...
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Reversible Cryo-arrests of Living Cells to Pause Molecular Movements for High-resolution Imaging 采用活细胞可逆性冷冻停滞法暂停分子运动以进行高分辨率成像 作者:Jan Huebinger, Martin E. Masip, Jens Christmann, Frank Wehner and Philippe I. H. Bastiaens日期:04/20/2017,浏览量:5953,Q&A: 0
Fluorescence live-cell imaging by single molecule localization microscopy (SMLM) or fluorescence lifetime imaging microscopy (FLIM) in principle allows for the spatio-temporal observation of molecular patterns in individual, living cells. However, the dynamics of molecules within cells hamper their precise observation. We present here a detailed ...
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发育生物学

Physical Removal of the Midbody Remnant from Polarised Epithelial Cells Using Take-Up by Suction Pressure (TUSP) 采用吸压抽吸法(TUSP)物理去除极化上皮细胞中的中间体残留物 作者:Miguel Bernabé-Rubio, David C. Gershlick and Miguel A. Alonso日期:04/20/2017,浏览量:6866,Q&A: 0
In polarised epithelial cells the midbody forms at the apical cell surface during cytokinesis. Once severed, the midbody is inherited by one of the daughter cells remaining tethered to the apical plasma membrane where it participates in non-cytokinetic processes, such as primary ciliogenesis. Here, we describe a novel method to physically remove ...
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免疫学

Lentiviral Barcode Labeling and Transplantation of Fetal Liver Hematopoietic Stem and Progenitor Cells 胎儿肝脏造血干细胞和祖细胞的慢病毒条形码标记和移植 作者:Trine A. Kristiansen, Alexander Doyle and Joan Yuan日期:04/20/2017,浏览量:7215,Q&A: 0
Cellular barcoding enables the dissection of clonal dynamics in heterogeneous cell populations through single cell lineage tracing. The labeling of hematopoietic stem and progenitor cells (HSPCs) with unique and heritable DNA barcodes, makes it possible to resolve donor cell heterogeneity in terms of differentiation potential and lineage bias at ...
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微生物学

Isolation of the Dot/Icm Type IV Secretion System Core Complex from Legionella pneumophila for Negative Stain Electron Microscopy Studies 从嗜肺军团杆菌中分离适于电子显微镜负染色技术检测的Dot/Icm IV型分泌系统核心复合物 作者:Tomoko Kubori and Hiroki Nagai日期:04/20/2017,浏览量:6267,Q&A: 0
Legionella possesses a pivotal secretion machinery to deliver virulence factors to eukaryotic host cells. In this protocol, we describe the procedure for isolation of the native core complex of the Dot/Icm type IV secretion system from L. pneumophila aiming to perform biochemical and transmission electron microscopy analyses.
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CRISPR/Cas9 Editing of the Bacillus subtilis Genome 枯草芽孢杆菌基因组的CRISPR/Cas9编辑 作者:Peter E. Burby and Lyle A. Simmons日期:04/20/2017,浏览量:15884,Q&A: 4
A fundamental procedure for most modern biologists is the genetic manipulation of the organism under study. Although many different methods for editing bacterial genomes have been used in laboratories for decades, the adaptation of CRISPR/Cas9 technology to bacterial genetics has allowed researchers to manipulate bacterial genomes with ...
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分子生物学

Expression and Analysis of Flow-regulated Ion Channels in Xenopus Oocytes 在非洲爪蟾卵母细胞中表达分析流量调节型的离子通道 作者:Shujie Shi and Marcelo D. Carattino日期:04/20/2017,浏览量:7608,Q&A: 0
Mechanically-gated ion channels play key roles in mechanotransduction, a process that translates physical forces into biological signals. Epithelial and endothelial cells are exposed to laminar shear stress (LSS), a tangential force exerted by flowing fluids against the wall of vessels and epithelia. The protocol outlined herein has been used to ...
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RNA Degradation Assay Using RNA Exosome Complexes, Affinity-purified from HEK-293 Cells 使用从HEK-293细胞亲和纯化获得的RNA外泌体复合物进行RNA降解测定 作者:Michal Domanski and John LaCava日期:04/20/2017,浏览量:7119,Q&A: 0
The RNA exosome complex plays a central role in RNA processing and regulated turnover. Present both in cytoplasm and nucleus, the exosome functions through associations with ribonucleases and various adapter proteins (reviewed in [Kilchert et al., 2016]). The RNA exosome-associated EXOSC10 protein is a distributive, 3’-5’ exoribonuclease. ...
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神经科学

The Object Context-place-location Paradigm for Testing Spatial Memory in Mice 用于测试小鼠空间记忆对象环境-地点-位置范例 作者:Edith Lesburguères, Panayiotis Tsokas, Todd Charlton Sacktor and André Antonio Fenton日期:04/20/2017,浏览量:9644,Q&A: 0
This protocol was originally designed to examine long-term spatial memory in PKMζ knockout (i.e., PKMζ-null) mice (Tsokas et al., 2016). Our main goal was to test whether the ability of these animals to maintain previously acquired spatial information was sensitive to the type and complexity of the spatial information that needs ...
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Evaluation of Muscle Performance in Mice by Treadmill Exhaustion Test and Whole-limb Grip Strength Assay 通过跑步机疲劳测试和全肢握力测定评估小鼠肌肉性能 作者:Beatriz Castro and Shihuan Kuang日期:04/20/2017,浏览量:11702,Q&A: 0
In vivo muscle function testing has become of great interest as primary phenotypic analysis of muscle performance. This protocol provides detailed procedures to perform the treadmill exhaustion test and the whole-limb grip strength assay, two methods commonly used in the neuromuscular research field.
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Preparation of Primary Astrocyte Culture Derived from Human Glioblastoma Multiforme Specimen 源自人多形性胶质母细胞瘤标本的原发性星形细胞培养物的制备 作者:Mansoureh Hashemi and Mahmoudreza Hadjighassem日期:04/20/2017,浏览量:8521,Q&A: 0
Glioblastoma multiforme (GBM) is a grade 4 astrocytoma tumor in central nervous system. Astrocytes can be isolated from human GBM. Study of astrocytes can provide insights about the formation, progression and recurrence of glioblastoma. For using isolated astrocytes, new studies can be designed in the fields of pharmacology, neuroscience and ...
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植物科学

Forest GPP Calculation Using Sap Flow and Water Use Efficiency Measurements 利用液流和水分利用效率测算森林的GPP 作者:Fyodor Tatarinov, Eyal Rotenberg, Dan Yakir and Tamir Klein日期:04/20/2017,浏览量:6672,Q&A: 0
This is a protocol to evaluate gross primary productivity (GPP) of a forest stand based on the measurements of tree’s sap flow (SF), 13C derived water use efficiency (WUE), and meteorological (met) data. GPP was calculated from WUE and stomatal conductance (gs), the later obtained from SF up-scaled from sampled trees to stand ...
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Measurement of Stomatal Conductance in Rice 水稻气孔导度测量 作者:Yin Wang and Toshinori Kinoshita日期:04/20/2017,浏览量:10448,Q&A: 0
Stomatal conductance, the reciprocal of stomatal resistance, represents the gas exchange ability of stomata. Generally, the stomatal conductance is higher when stomata open wider, and vice versa. In this protocol, we describe how to measure stomatal conductance in rice using Li-6400 (Licor, USA).
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Rapid Isolation of Total Protein from Arabidopsis Pollen 从拟南芥花粉中快速分离总蛋白 作者:Ming Chang and Shanjin Huang日期:04/20/2017,浏览量:8308,Q&A: 0
Arabidopsis pollen is an excellent system for answering important biological questions about the establishment and maintenance of cellular polarity and polar cell growth, because these processes are amenable to the genetic and genomic approaches that are readily available in Arabidopsis. Given that proteins are the direct ...
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Single Molecule RNA FISH in Arabidopsis Root Cells 拟南芥根细胞中的单个RNA分子FISH 作者:Susan Duncan, Tjelvar S. G. Olsson, Matthew Hartley, Caroline Dean and Stefanie Rosa日期:04/20/2017,浏览量:11690,Q&A: 0
Methods that allow the study of gene expression regulation are continually advancing. Here, we present an in situ hybridization protocol capable of detecting individual mRNA molecules in plant root cells, thus permitting the accurate quantification and localization of mRNA within fixed samples (Duncan et al., 2016; Rosa et al ...
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Growth Assay for the Stem Parasitic Plants of the Genus Cuscuta 菟丝子属茎寄生植物的生长测定 作者:Volker Hegenauer , Max Welz, Max Körner and Markus Albert日期:04/20/2017,浏览量:9773,Q&A: 0
Cuscuta spp. are widespread obligate holoparasitic plants with a broad host spectrum. Rootless Cuscuta penetrates host stems with so called haustoria to form a direct connection to the host vascular tissue (Dawson et al., 1994; Lanini and Kogan, 2005; Kaiser et al., 2015). This connection allows a steady uptake ...
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Automated Tracking of Root for Confocal Time-lapse Imaging of Cellular Processes 对根进行自动跟踪以进行细胞过程的共焦延时成像 作者:Mehdi Doumane, Claire Lionnet, Vincent Bayle, Yvon Jaillais and Marie-Cécile Caillaud日期:04/20/2017,浏览量:8581,Q&A: 0
Here we describe a protocol that enables to automatically perform time-lapse imaging of growing root tips for several hours. Plants roots expressing fluorescent proteins or stained with dyes are imaged while they grow using automatic movement of the microscope stage that compensates for root growth and allows to follow a given region of the root ...
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干细胞

Mimicking Angiogenesis in vitro: Three-dimensional Co-culture of Vascular Endothelial Cells and Perivascular Cells in Collagen Type I Gels 体外模拟血管发生:血管内皮细胞和血管周围细胞在I型胶原蛋白凝胶中的三维共培养 作者:Markus Auler, Lena Pitzler, Ernst Pöschl, Zhigang Zhou and Bent Brachvogel日期:04/20/2017,浏览量:14454,Q&A: 0
Angiogenesis defines the process of formation of new vascular structures form existing blood vessels, involved during development, repair processes like wound healing but also linked to pathological changes. During angiogenic processes, endothelial cells build a vascular network and recruit perivascular cells to form mature, stable vessels. ...
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