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  • Multiplexed proteome analysis with neutron-encoded stable isotope labeling in cells and mice
    Nat. Protoc. (IF 10.032) Pub Date : 2018-01-11
    Katherine A Overmyer, Stefka Tyanova, Alex S Hebert, Michael S Westphall, Jürgen Cox, Joshua J Coon

    We describe a protocol for multiplexed proteomic analysis using neutron-encoded (NeuCode) stable isotope labeling of amino acids in cells (SILAC) or mice (SILAM). This method currently enables simultaneous comparison of up to nine treatment and control proteomes. Another important advantage over traditional SILAC/SILAM is that shorter labeling times are required. Exploiting the small mass differences that correspond to subtle differences in the neutron-binding energies of different isotopes, the amino acids used in NeuCode SILAC/SILAM differ in mass by just a few milliDaltons. Isotopologs of lysine are introduced into cells or mammals, via the culture medium or diet, respectively, to metabolically label the proteome. Labeling time is ∼2 weeks for cultured cells and 3–4 weeks for mammals. The proteins are then extracted, relevant samples are combined, and these are enzymatically digested with lysyl endopeptidase (Lys-C). The resultant peptides are chromatographically separated and then mass analyzed. During mass spectrometry (MS) data acquisition, high-resolution MS1 spectra (≥240,000 resolving power at m/z = 400) reveal the embedded isotopic signatures, enabling relative quantification, while tandem mass spectra, collected at lower resolutions, provide peptide identities. Both types of spectra are processed using NeuCode-enabled MaxQuant software. In total, the approximate completion time for the protocol is 3–5 weeks.

    更新日期:2018-01-11
  • CRISPR-Cas9-based genome-wide screening of Toxoplasma gondii
    Nat. Protoc. (IF 10.032) Pub Date : 2018-01-11
    Saima M Sidik, Diego Huet, Sebastian Lourido

    Apicomplexan parasites, such as Toxoplasma gondii, cause extensive morbidity and mortality in humans and livestock, highlighting the need for a deeper understanding of their molecular biology. Although techniques for the generation of targeted gene disruptions have long been available for apicomplexans, such methods are not readily scalable to the entire genome. We recently used CRISPR-Cas9 to disrupt all nuclear protein–coding genes in T. gondii using a pooled format. The method relies on transfection of a guide RNA library into parasites constitutively expressing Cas9. Here, we present the complete workflow of such a screen, including preparation of the guide RNA library, growth and testing of the recipient strain, generation of the mutant population, culture conditions for the screen, preparation of genomic DNA libraries, next-generation sequencing of the guide RNA loci, and analysis to detect fitness-conferring genes. This method can be deployed to study how culture conditions affect the repertoire of genes needed by parasites, which will enable studies of their metabolic needs, host specificity, and drug-resistance mechanisms. In addition, by manipulating the background in which the screen is performed, researchers will be able to investigate genetic interactions, which may help uncover redundancy or epistasis in the parasite genome. Using this method, a genome-wide screen and its analysis can be completed in 3 weeks, after ∼1 month of preparation to generate the library and grow the cells needed, making it a powerful tool for uncovering functionally important genes in apicomplexan parasites.

    更新日期:2018-01-11
  • On-demand synthesis of organozinc halides under continuous flow conditions
    Nat. Protoc. (IF 10.032) Pub Date : 2018-01-11
    Mateo Berton, Lena Huck, Jesús Alcázar

    Organozinc reagents are versatile building blocks for introducing C(sp2)-C(sp3) and C(sp3)-C(sp3) bonds into organic structures. However, despite their ample synthetic versatility and broad functional group tolerance, the use of organozinc reagents in the laboratory is limited because of their instability, exothermicity and water sensitivity, as well as their labor-intensive preparation. Herein, we describe an on-demand synthesis of these useful reagents under continuous flow conditions, overcoming these primary limitations and supporting widespread adoption of these reagents in synthetic organic chemistry. To exemplify this procedure, a solution of ethyl zincbromoacetate is prepared by flowing ethyl bromoacetate through a column containing metallic zinc. The temperature of the column is controlled by a heating jacket and a thermocouple in close contact with it. Advice on how to perform the procedure using alternative equipment is also given to allow a wider access to the methodology. Here we describe the preparation of 50 ml of solution, which takes 1 h 40 min, although up to 250–300 ml can be prepared with the same column setup at a rate of 30 ml per h. The procedure provides the reagent as a clean solution with reproducible concentration. Organozinc solutions generated in flow can be coupled to a second flow reactor to perform a Reformatsky reaction or can be collected over a flask containing the required reagents for a batch Negishi reaction.

    更新日期:2018-01-11
  • Colonoscopy-based colorectal cancer modeling in mice with CRISPR–Cas9 genome editing and organoid transplantation
    Nat. Protoc. (IF 10.032) Pub Date : 2018-01-04
    Jatin Roper, Tuomas Tammela, Adam Akkad, Mohammad Almeqdadi, Sebastian B Santos, Tyler Jacks, Ömer H Yilmaz

    Most genetically engineered mouse models (GEMMs) of colorectal cancer are limited by tumor formation in the small intestine, a high tumor burden that limits metastasis, and the need to generate and cross mutant mice. Cell line or organoid transplantation models generally produce tumors in ectopic locations—such as the subcutaneous space, kidney capsule, or cecal wall—that do not reflect the native stromal environment of the colon mucosa. Here, we describe detailed protocols to rapidly and efficiently induce site-directed tumors in the distal colon of mice that are based on colonoscopy-guided mucosal injection. These techniques can be adapted to deliver viral vectors carrying Cre recombinase, CRISPR–Cas9 components, CRISPR-engineered mouse tumor organoids, or human cancer organoids to mice to model the adenoma–carcinoma–metastasis sequence of tumor progression. The colonoscopy injection procedure takes ∼15 min, including preparation. In our experience, anyone with reasonable hand–eye coordination can become proficient with mouse colonoscopy and mucosal injection with a few hours of practice. These approaches are ideal for a wide range of applications, including assessment of gene function in tumorigenesis, examination of tumor–stroma interactions, studies of cancer metastasis, and translational research with patient-derived cancers.

    更新日期:2018-01-04
  • A surgical orthotopic organoid transplantation approach in mice to visualize and study colorectal cancer progression
    Nat. Protoc. (IF 10.032) Pub Date : 2018-01-04
    Arianna Fumagalli, Saskia J E Suijkerbuijk, Harry Begthel, Evelyne Beerling, Koen C Oost, Hugo J Snippert, Jacco van Rheenen, Jarno Drost

    Most currently available colorectal cancer (CRC) mouse models are not suitable for studying progression toward the metastatic stage. Recently, establishment of tumor organoid lines, either from murine CRC models or patients, and the possibility of engineering them with genome-editing technologies, have provided a large collection of tumor material faithfully recapitulating phenotypic and genetic heterogeneity of native tumors. To study tumor progression in the natural in vivo environment, we developed an orthotopic approach based on transplantation of CRC organoids into the cecal epithelium. The 20-min procedure is described in detail here and enables growth of transplanted organoids into a single tumor mass within the intestinal tract. Due to long latency, tumor cells are capable of spreading through the blood circulation and forming metastases at distant sites. This method is designed to generate tumors suitable for studying CRC progression, thereby providing the opportunity to visualize tumor cell dynamics in vivo in real time by intravital microscopy.

    更新日期:2018-01-04
  • Use of a three-layer gradient system of cells for rat testicular organoid generation
    Nat. Protoc. (IF 10.032) Pub Date : 2018-01-04
    João Pedro Alves-Lopes, Olle Söder, Jan-Bernd Stukenborg

    We have recently developed a 3D culture system that allows the reorganization of rat primary testicular cells into organoids with a functional blood–testis barrier, as well as the establishment and maintenance of germ cells. The innovative aspect of our model, the three-layer gradient system (3-LGS), comprises cells combined with Matrigel placed between two layers of Matrigel without cells, which creates a gradient of cells and allows the reorganization of testicular cells into organized structures after 5–7 d in culture. This reorganization is not observed when testicular cells are suspended in only one layer of Matrigel, the methodology used in the majority of the protocols for generating organoids. The model can be applied to follow and quantify cell migration during testicular organoid formation, and to explore the role of growth factors and the toxic effects of drugs and environmental contaminants on germ cell maintenance and blood–testis barrier integrity. The 3-LGS is a robust and reproducible method that requires a small volume of Matrigel and a low number of cells (16 μl and 132,000 cells, respectively), enabling and facilitating high-throughput analysis of germ-to-somatic cell associations in vitro.

    更新日期:2018-01-04
  • High-throughput in situ X-ray screening of and data collection from protein crystals at room temperature and under cryogenic conditions
    Nat. Protoc. (IF 10.032) Pub Date : 2018-01-04
    Jana Broecker, Takefumi Morizumi, Wei-Lin Ou, Viviane Klingel, Anling Kuo, David J Kissick, Andrii Ishchenko, Ming-Yue Lee, Shenglan Xu, Oleg Makarov, Vadim Cherezov, Craig M Ogata, Oliver P Ernst

    Protein crystallography has significantly advanced in recent years, with in situ data collection, in which crystals are placed in the X-ray beam within their growth medium, being a major point of focus. In situ methods eliminate the need to harvest crystals, a previously unavoidable drawback, particularly for often small membrane-protein crystals. Here, we present a protocol for the high-throughput in situ X-ray screening of and data collection from soluble and membrane-protein crystals at room temperature (20–25°C) and under cryogenic conditions. The Mylar in situ method uses Mylar-based film sandwich plates that are inexpensive, easy to make, and compatible with automated imaging, and that show very low background scattering. They support crystallization in microbatch and vapor-diffusion modes, as well as in lipidic cubic phases (LCPs). A set of 3D-printed holders for differently sized patches of Mylar sandwich films makes the method robust and versatile, allows for storage and shipping of crystals, and enables automated mounting at synchrotrons, as well as goniometer-based screening and data collection. The protocol covers preparation of in situ plates and setup of crystallization trials; 3D printing and assembly of holders; opening of plates, isolation of film patches containing crystals, and loading them onto holders; basic screening and data-collection guidelines; and unloading of holders, as well as reuse and recycling of them. In situ plates are prepared and assembled in 1 h; holders are 3D-printed and assembled in ≤90 min; and an in situ plate is opened, and a film patch containing crystals is isolated and loaded onto a holder in 5 min.

    更新日期:2018-01-04
  • 3D molecular cartography using LC–MS facilitated by Optimus and 'ili software
    Nat. Protoc. (IF 10.032) Pub Date : 2017-12-21
    Ivan Protsyuk, Alexey V Melnik, Louis-Felix Nothias, Luca Rappez, Prasad Phapale, Alexander A Aksenov, Amina Bouslimani, Sergey Ryazanov, Pieter C Dorrestein, Theodore Alexandrov

    Our skin, our belongings, the world surrounding us, and the environment we live in are covered with molecular traces. Detecting and characterizing these molecular traces is necessary to understand the environmental impact on human health and disease, and to decipher complex molecular interactions between humans and other species, particularly microbiota. We recently introduced 3D molecular cartography for mapping small organic molecules (including metabolites, lipids, and environmental molecules) found on various surfaces, including the human body. Here, we provide a protocol and open-source software for 3D molecular cartography. The protocol includes step-by-step procedures for sample collection and processing, liquid chromatography–mass spectrometry (LC–MS)-based metabolomics, quality control (QC), molecular identification using MS/MS, data processing, and visualization with 3D models of the sampled environment. The LC–MS method was optimized for a broad range of small organic molecules. We enable scientists to reproduce our previously obtained results, and illustrate the broad utility of our approach with molecular maps of a rosemary plant and an ATM keypad after a PIN code was entered. To promote reproducibility, we introduce cartographical snapshots: files that describe a particular map and visualization settings, and that can be shared and loaded to reproduce the visualization. The protocol enables molecular cartography to be performed in any mass spectrometry laboratory and, in principle, for any spatially mapped data. We anticipate applications, in particular, in medicine, ecology, agriculture, biotechnology, and forensics. The protocol takes 78 h for a molecular map of 100 spots, excluding the reagent setup.

    更新日期:2017-12-21
  • Live-cell measurements of kinase activity in single cells using translocation reporters
    Nat. Protoc. (IF 10.032) Pub Date : 2017-12-21
    Takamasa Kudo, Stevan Jeknić, Derek N Macklin, Sajia Akhter, Jacob J Hughey, Sergi Regot, Markus W Covert

    Although kinases are important regulators of many cellular processes, measuring their activity in live cells remains challenging. We have developed kinase translocation reporters (KTRs), which enable multiplexed measurements of the dynamics of kinase activity at a single-cell level. These KTRs are composed of an engineered construct in which a kinase substrate is fused to a bipartite nuclear localization signal (bNLS) and nuclear export signal (NES), as well as to a fluorescent protein for microscopy-based detection of its localization. The negative charge introduced by phosphorylation of the substrate is used to directly modulate nuclear import and export, thereby regulating the reporter's distribution between the cytoplasm and nucleus. The relative cytoplasmic versus nuclear fluorescence of the KTR construct (the C/N ratio) is used as a proxy for the kinase activity in living, single cells. Multiple KTRs can be studied in the same cell by fusing them to different fluorescent proteins. Here, we present a protocol to execute and analyze live-cell microscopy experiments using KTRs. We describe strategies for development of new KTRs and procedures for lentiviral expression of KTRs in a cell line of choice. Cells are then plated in a 96-well plate, from which multichannel fluorescent images are acquired with automated time-lapse microscopy. We provide detailed guidance for a computational analysis and parameterization pipeline. The entire procedure, from virus production to data analysis, can be completed in ∼10 d.

    更新日期:2017-12-21
  • Single-cell microscopy of suspension cultures using a microfluidics-assisted cell screening platform
    Nat. Protoc. (IF 10.032) Pub Date : 2017-12-21
    Burak Okumus, Charles J Baker, Juan Carlos Arias-Castro, Ghee Chuan Lai, Emanuele Leoncini, Somenath Bakshi, Scott Luro, Dirk Landgraf, Johan Paulsson

    Studies that rely on fluorescence imaging of nonadherent cells that are cultured in suspension, such as Escherichia coli, are often hampered by trade-offs that must be made between data throughput and imaging resolution. We developed a platform for microfluidics-assisted cell screening (MACS) that overcomes this trade-off by temporarily immobilizing suspension cells within a microfluidics chip. This enables high-throughput and automated single-cell microscopy for a wide range of cell types and sizes. As cells can be rapidly sampled directly from a suspension culture, MACS bypasses the need for sample preparation, and therefore allows measurements without perturbing the native cell physiology. The setup can also be integrated with complex growth chambers, and can be used to enrich or sort the imaged cells. Furthermore, MACS facilitates the visualization of individual cytoplasmic fluorescent proteins (FPs) in E. coli, allowing low-abundance proteins to be counted using standard total internal reflection fluorescence (TIRF) microscopy. Finally, MACS can be used to impart mechanical pressure for assessing the structural integrity of individual cells and their response to mechanical perturbations, or to make cells take up chemicals that otherwise would not pass through the membrane. This protocol describes the assembly of electronic control circuitry, the construction of liquid-handling components and the creation of the MACS microfluidics chip. The operation of MACS is described, and automation software is provided to integrate MACS control with image acquisition. Finally, we provide instructions for extending MACS using an external growth chamber (1 d) and for how to sort rare cells of interest.

    更新日期:2017-12-21
  • Easi-CRISPR for creating knock-in and conditional knockout mouse models using long ssDNA donors
    Nat. Protoc. (IF 10.032) Pub Date : 2017-12-21
    Hiromi Miura, Rolen M Quadros, Channabasavaiah B Gurumurthy, Masato Ohtsuka

    CRISPR/Cas9-based genome editing can easily generate knockout mouse models by disrupting the gene sequence, but its efficiency for creating models that require either insertion of exogenous DNA (knock-in) or replacement of genomic segments is very poor. The majority of mouse models used in research involve knock-in (reporters or recombinases) or gene replacement (e.g., conditional knockout alleles containing exons flanked by LoxP sites). A few methods for creating such models have been reported that use double-stranded DNA as donors, but their efficiency is typically 1–10% and therefore not suitable for routine use. We recently demonstrated that long single-stranded DNAs (ssDNAs) serve as very efficient donors, both for insertion and for gene replacement. We call this method efficient additions with ssDNA inserts–CRISPR (Easi-CRISPR) because it is a highly efficient technology (efficiency is typically 30–60% and reaches as high as 100% in some cases). The protocol takes ∼2 months to generate the founder mice.

    更新日期:2017-12-21
  • Transient expression of human antibodies in mammalian cells
    Nat. Protoc. (IF 10.032) Pub Date : 2017-12-14
    Rodrigo Vazquez-Lombardi, Damien Nevoltris, Ansha Luthra, Peter Schofield, Carsten Zimmermann, Daniel Christ

    Mammalian cells are powerful expression systems for producing glycosylated recombinant antibody preparations with minimal endotoxin contamination. This protocol describes procedures for antibody design, expression, purification and characterization.

    更新日期:2017-12-15
  • Use of TAI-FISH to visualize neural ensembles activated by multiple stimuli
    Nat. Protoc. (IF 10.032) Pub Date : 2017-12-14
    Qi Zhang, Qiye He, Jihua Wang, Chaoying Fu, Hailan Hu

    This protocol describes a dual mRNA and protein labeling strategy that allows identification of activated neuronal assemblies in response to two temporally separated stimuli in mouse brain sections.

    更新日期:2017-12-15
  • Mapping the small RNA interactome in bacteria using RIL-seq
    Nat. Protoc. (IF 10.032) Pub Date : 2017-12-07
    Sahar Melamed, Raya Faigenbaum-Romm, Asaf Peer, Niv Reiss, Omer Shechter, Amir Bar, Yael Altuvia, Liron Argaman, Hanah Margalit

    This protocol describes an experimental–computational methodology for mapping the small RNA interactome in bacteria.

    更新日期:2017-12-13
  • Expansion of patient-derived circulating tumor cells from liquid biopsies using a CTC microfluidic culture device
    Nat. Protoc. (IF 10.032) Pub Date : 2017-12-07
    Bee Luan Khoo, Gianluca Grenci, Ying Bena Lim, Soo Chin Lee, Jongyoon Han, Chwee Teck Lim

    This protocol describes a microfluidics approach for culturing liquid-biopsy-derived circulating tumor cell clusters to predict a patient's response toward various therapeutic strategies.

    更新日期:2017-12-13
  • Assembly of phospholipid nanodiscs of controlled size for structural studies of membrane proteins by NMR
    Nat. Protoc. (IF 10.032) Pub Date : 2017-12-07
    Franz Hagn, Mahmoud L Nasr, Gerhard Wagner

    The applications of solution-state NMR of membrane proteins are often limited by difficulty in finding a suitable membrane mimetic of tailored size that shows native-like membrane properties and provides long-term stability. This protocol describes how to assemble phospholipid nanodiscs and incorporate membrane proteins for NMR-structural studies.

    更新日期:2017-12-13
  • Measuring mutation accumulation in single human adult stem cells by whole-genome sequencing of organoid cultures
    Nat. Protoc. (IF 10.032) Pub Date : 2017-12-07
    Myrthe Jager, Francis Blokzijl, Valentina Sasselli, Sander Boymans, Roel Janssen, Nicolle Besselink, Hans Clevers, Ruben van Boxtel, Edwin Cuppen

    This protocol describes a method for cataloging genome-wide mutations that accumulated during life or culture in single adult stem cells of different human tissues, by combining whole-genome sequencing with organoid-culture technologies.

    更新日期:2017-12-13
  • Langmuir–Blodgett nanotemplates for protein crystallography
    Nat. Protoc. (IF 10.032) Pub Date : 2017-11-30
    Eugenia Pechkova, Claudio Nicolini

    Protein crystallization still presents a challenge for X-ray crystallography. This protocol describes the Langmuir–Blodgett nanotemplate method, in which 2D protein LB nanotemplates trigger formation of 3D protein crystals by hanging-drop vapor diffusion.

    更新日期:2017-11-30
  • Visualizing endocytic recycling and trafficking in live neurons by subdiffractional tracking of internalized molecules
    Nat. Protoc. (IF 10.032) Pub Date : 2017-11-30
    Merja Joensuu, Ramon Martínez-Mármol, Pranesh Padmanabhan, Nick R Glass, Nela Durisic, Matthew Pelekanos, Mahdie Mollazade, Giuseppe Balistreri, Rumelo Amor, Justin J Cooper-White, Geoffrey J Goodhill, Frédéric A Meunier

    This protocol describes a pulse–chase approach to studying activity-dependent internalization of fluorescent ligands into endocytic compartments using subdiffractional single-particle tracking in live hippocampal neurons.

    更新日期:2017-11-30
  • Preparation of viable adult ventricular myocardial slices from large and small mammals
    Nat. Protoc. (IF 10.032) Pub Date : 2017-11-30
    Samuel A Watson, Martina Scigliano, Ifigeneia Bardi, Raimondo Ascione, Cesare M Terracciano, Filippo Perbellini

    This protocol describes how to obtain 100- to 400-μm-thick slices of a living myocardium from rodents, pigs, humans and dogs that retain the native multicellularity, architecture and physiology of the heart.

    更新日期:2017-11-30
  • Deriving genotypes from RAD-seq short-read data using Stacks
    Nat. Protoc. (IF 10.032) Pub Date : 2017-11-30
    Nicolas C Rochette, Julian M Catchen

    In this protocol, the authors provide a strategy and set of methods to analyze restriction-site-associated DNA-sequencing (RAD-seq) data using Stacks, enabling the genome-wide discovery and genotyping of SNPs across a range of systems.

    更新日期:2017-11-30
  • Production of knock-in mice in a single generation from embryonic stem cells
    Nat. Protoc. (IF 10.032) Pub Date : 2017-11-16
    Hideki Ukai, Hiroshi Kiyonari, Hiroki R Ueda

    This protocol describes the generation of mice entirely derived from genome-edited embryonic stem cells, enabling the production of transgenic mice in a single generation.

    更新日期:2017-11-17
  • Multimodal profiling of single-cell morphology, electrophysiology, and gene expression using Patch-seq
    Nat. Protoc. (IF 10.032) Pub Date : 2017-11-16
    Cathryn R Cadwell, Federico Scala, Shuang Li, Giulia Livrizzi, Shan Shen, Rickard Sandberg, Xiaolong Jiang, Andreas S Tolias

    This protocol describes how to integrate whole-cell patch-clamp in single neurons from mouse brain tissue slices with single-cell RNA sequencing and morphological recovery.

    更新日期:2017-11-17
  • Chemical synthesis of membrane proteins by the removable backbone modification method
    Nat. Protoc. (IF 10.032) Pub Date : 2017-11-16
    Shan Tang, Chao Zuo, Dong-Liang Huang, Xiao-Ying Cai, Long-Hua Zhang, Chang-Lin Tian, Ji-Shen Zheng, Lei Liu

    This protocol describes how to chemically synthesize membrane proteins through the installation of solubilizing removable backbone modification tags into hydrophobic transmembrane peptides. The implementation of the protocol is demonstrated by the chemical synthesis of phosphorylated M2 (M2-pSer64), a 97-aa proton channel protein from the influenza A virus. The synthesis of M2-pSer64 at milligram scale takes ∼200 working hours (excluding the time for lyophilizations).

    更新日期:2017-11-17
  • Compartmentalized partnered replication for the directed evolution of genetic parts and circuits
    Nat. Protoc. (IF 10.032) Pub Date : 2017-11-09
    Zhanar Abil, Jared W Ellefson, Jimmy D Gollihar, Ella Watkins, Andrew D Ellington

    This protocol describes the procedures for compartmentalized partnered replication (CPR), an emulsion-based directed evolution method for the generation of proteins, genetic elements, and genetic circuits with improved or altered function.

    更新日期:2017-11-10
  • Chromatin-state discovery and genome annotation with ChromHMM
    Nat. Protoc. (IF 10.032) Pub Date : 2017-11-09
    Jason Ernst, Manolis Kellis

    This protocol describes how to use ChromHMM, a robust open-source software package that enables the learning of chromatin states, annotates their occurrences across the genome, and facilitates their biological interpretation.

    更新日期:2017-11-10
  • Identification of RNA-binding domains of RNA-binding proteins in cultured cells on a system-wide scale with RBDmap
    Nat. Protoc. (IF 10.032) Pub Date : 2017-11-02
    Alfredo Castello, Christian K. Frese, Bernd Fischer, Aino I Järvelin, Rastislav Horos, Anne-Marie Alleaume, Sophia Foehr, Tomaz Curk, Jeroen Krijgsveld, Matthias W Hentze

    Here the authors provide an extension to their earlier RNA interactome capture protocol. This Protocol Extension describes RBDmap—a method to identify the regions of RNA-binding proteins engaged in native interactions with RNA, in a proteome-wide manner.

    更新日期:2017-11-02
  • A high-throughput in vivo screening method in the mouse for identifying regulators of metastatic colonization
    Nat. Protoc. (IF 10.032) Pub Date : 2017-11-02
    Anneliese O Speak, Agnieszka Swiatkowska, Natasha A Karp, Mark J Arends, David J Adams, Louise van der Weyden

    In this protocol, the authors present an experimental metastasis assay in which cancer cells are injected into the tail vein of a mouse, and the resulting secondary organ colonization is assessed, primarily in the lung, 10 d later.

    更新日期:2017-11-02
  • The assembly and use of continuous flow systems for chemical synthesis
    Nat. Protoc. (IF 10.032) Pub Date : 2017-10-26
    Joshua Britton, Timothy F Jamison

    Flow chemistry is an attractive alternative to batch chemistry in cases in which improved safety and reaction efficiency can be achieved. This protocol describes the assembly of a continuous flow apparatus from readily available and affordable parts.

    更新日期:2017-10-30
  • Preparation of glycoconjugates from unprotected carbohydrates for protein-binding studies
    Nat. Protoc. (IF 10.032) Pub Date : 2017-10-26
    Christian T Hjuler, Nicolai N Maolanon, Jørgen Sauer, Jens Stougaard, Mikkel B Thygesen, Knud J Jensen

    Conjugation of oligosaccharides to probes and surfaces is useful in the development of biochemical assays to assess carbohydrate–protein interactions. In this protocol, conjugation is enabled by using a bifunctional oligo(ethylene glycol) linker.

    更新日期:2017-10-30
  • Measuring protein structural changes on a proteome-wide scale using limited proteolysis-coupled mass spectrometry
    Nat. Protoc. (IF 10.032) Pub Date : 2017-10-26
    Simone Schopper, Abdullah Kahraman, Pascal Leuenberger, Yuehan Feng, Ilaria Piazza, Oliver Müller, Paul J Boersema, Paola Picotti

    Many intra- and extracellular signals induce structural changes in proteins. Schopper et al., describe a limited proteolysis–based mass spectrometry (LiP-MS) approach to characterizing these changes at a proteome-wide scale.

    更新日期:2017-10-30
  • Analysis of human cerebrospinal fluid monoamines and their cofactors by HPLC
    Nat. Protoc. (IF 10.032) Pub Date : 2017-10-19
    Marta Batllori, Marta Molero-Luis, Aida Ormazabal, Mercedes Casado, Cristina Sierra, Angels García-Cazorla, Manju Kurian, Simon Pope, Simon J Heales, Rafael Artuch

    The levels of monoamines and their cofactors in cerebrospinal fluid are strong indicators for dopamine and serotonin biosynthesis and turnover. This protocol describes a set of HPLC-based approaches for the quantitative detection of these molecules.

    更新日期:2017-10-19
  • Identification of cross talk between SUMOylation and ubiquitylation using a sequential peptide immunopurification approach
    Nat. Protoc. (IF 10.032) Pub Date : 2017-10-19
    Francis P McManus, Frédéric Lamoliatte, Pierre Thibault

    Recent studies have uncovered substantial cross talk between the ubiquitylation and SUMOylation pathways. Using sequential affinity purification and mass spectrometry, this protocol enables the identification of proteins that are modified by both pathways.

    更新日期:2017-10-19
  • Cell-derived matrices for studying cell proliferation and directional migration in a complex 3D microenvironment
    Nat. Protoc. (IF 10.032) Pub Date : 2017-10-19
    Riina Kaukonen, Guillaume Jacquemet, Hellyeh Hamidi, Johanna Ivaska

    This protocol describes how to produce cell-derived matrices from fibroblasts. These matrices can be used to provide a 3D scaffold for cell culture and to investigate cell behavior in complex microenvironments.

    更新日期:2017-10-19
  • Facile synthesis of gold nanomaterials with unusual crystal structures
    Nat. Protoc. (IF 10.032) Pub Date : 2017-10-12
    Zhanxi Fan, Xiao Huang, Ye Chen, Wei Huang, Hua Zhang

    Crystal-phase-controlled synthesis of noble metal nanomaterials is a promising strategy to tune their physicochemical properties. Gold nanomaterials with unusual crystal structures (e.g., hcp, hcp/fcc and 4H) can be prepared under mild conditions.

    更新日期:2017-10-12
  • Simultaneous measurement of sleep and feeding in individual Drosophila
    Nat. Protoc. (IF 10.032) Pub Date : 2017-10-12
    Keith R Murphy, Jin Hong Park, Robert Huber, William W Ja

    This protocol describes the construction and use of the Activity Recording CAFE, an automated image-tracking-based system for the integrated measurement of sleep and feeding in individual Drosophila.

    更新日期:2017-10-12
  • Genome-wide mapping of DNase I hypersensitive sites in rare cell populations using single-cell DNase sequencing
    Nat. Protoc. (IF 10.032) Pub Date : 2017-10-12
    James Cooper, Yi Ding, Jiuzhou Song, Keji Zhao

    DNase I hypersensitive sites (DHSs) are regions of accessible chromatin that are indicative of regions involved in the regulation of gene expression. scDNase-seq allows genome-wide detection of DHSs from a low number of cells, including single cells.

    更新日期:2017-10-12
  • Synthesis and characterization of well-defined hydrogel matrices and their application to intestinal stem cell and organoid culture
    Nat. Protoc. (IF 10.032) Pub Date : 2017-10-05
    Nikolce Gjorevski, Matthias P Lutolf

    This protocol describes the synthesis and application of hydrogel matrices comprising a poly(ethylene glycol) backbone, functionalized with cell adhesion cues and laminin-111. Uses include expanding stem cells and differentiating them into organoids.

    更新日期:2017-10-11
  • A cerebellar window for intravital imaging of normal and disease states in mice
    Nat. Protoc. (IF 10.032) Pub Date : 2017-10-05
    Vasileios Askoxylakis, Mark Badeaux, Sylvie Roberge, Ana Batista, Ned Kirkpatrick, Matija Snuderl, Zohreh Amoozgar, Giorgio Seano, Gino B Ferraro, Sampurna Chatterjee, Lei Xu, Dai Fukumura, Dan G Duda, Rakesh K Jain

    In this protocol, the skull overlying the cerebellum is removed and a window is applied, enabling intravital imaging to provide a detailed characterization of dynamic processes in this region of the mouse brain.

    更新日期:2017-10-11
  • Total chemical synthesis of histones and their analogs, assisted by native chemical ligation and palladium complexes
    Nat. Protoc. (IF 10.032) Pub Date : 2017-10-05
    Suman Kumar Maity, Muhammad Jbara, Guy Mann, Guy Kamnesky, Ashraf Brik

    Chemical synthesis of proteins (e.g., histones) allows precise insertion of modified amino acids. This protocol uses palladium chemistry to remove protecting groups and a removable solubilizing tag for the synthesis of lipophilic peptide segments.

    更新日期:2017-10-11
  • Combining confocal and atomic force microscopy to quantify single-virus binding to mammalian cell surfaces
    Nat. Protoc. (IF 10.032) Pub Date : 2017-10-05
    Richard Newton, Martin Delguste, Melanie Koehler, Andra C Dumitru, Pawel R Laskowski, Daniel J Müller, David Alsteens

    This protocol describes how to combine confocal and atomic force microscopy (AFM) to study the interactions between single viruses and their cell-surface receptors on live cells.

    更新日期:2017-10-11
  • Base-resolution stratification of cancer mutations using functional variomics
    Nat. Protoc. (IF 10.032) Pub Date : 2017-10-05
    Song Yi, Ning-Ning Liu, Limei Hu, Hui Wang, Nidhi Sahni

    This massively parallel pipeline enables high-throughput generation and confirmation of variants by Gateway cloning, barcoding, and next-generation sequencing, and their stratification by multiplexed interaction profiling and experimental validation.

    更新日期:2017-10-11
  • Generation and use of a humanized bone-marrow-ossicle niche for hematopoietic xenotransplantation into mice
    Nat. Protoc. (IF 10.032) Pub Date : 2017-09-21
    Andreas Reinisch, David Cruz Hernandez, Katharina Schallmoser, Ravindra Majeti

    Humanized bone-marrow-ossicle niches are formed in mice via in situ differentiation of bone-marrow-derived mesenchymal stromal cells and can be used for transplantation of normal and malignant human hematopoietic cells.

    更新日期:2017-09-21
  • Genetically encoded releasable photo-cross-linking strategies for studying protein–protein interactions in living cells
    Nat. Protoc. (IF 10.032) Pub Date : 2017-09-21
    Yi Yang, Haiping Song, Dan He, Shuai Zhang, Shizhong Dai, Xiao Xie, Shixian Lin, Ziyang Hao, Huangtao Zheng, Peng R Chen

    This protocol describes strategies for the characterization of transient protein–protein interactions and their interaction interfaces via genetically encoded releasable photo-cross-linkers.

    更新日期:2017-09-21
  • Synthesis of a HyCoSuL peptide substrate library to dissect protease substrate specificity
    Nat. Protoc. (IF 10.032) Pub Date : 2017-09-21
    Marcin Poreba, Guy S Salvesen, Marcin Drag

    This protocol describes HyCoSuL, an approach that uses tetrapeptides containing natural and >100 unnatural amino acids to screen for protease substrate specificity and to engineer highly active and selective substrates and activity-based probes.

    更新日期:2017-09-21
  • Lab-scale production of anhydrous diazomethane using membrane separation technology
    Nat. Protoc. (IF 10.032) Pub Date : 2017-09-14
    Doris Dallinger, C Oliver Kappe

    Diazomethane is useful for inserting methyl or methylene groups in organic synthesis. Unfortunately, it is explosive. A tube-in-flask reactor where the Teflon AF-2400 tube allows only the diazomethane produced to enter the flask can be used to prepare it safely.

    更新日期:2017-09-14
  • Rapid curation of gene disruption collections using Knockout Sudoku
    Nat. Protoc. (IF 10.032) Pub Date : 2017-09-14
    Isao A Anzai, Lev Shaket, Oluwakemi Adesina, Michael Baym, Buz Barstow

    Knockout Sudoku allows construction of whole-genome knockout collections for a wide range of microorganisms at a lower cost and increased speed, using combinatorial pooling, next-generation sequencing, and a Bayesian inference algorithm to process and annotate extremely large progenitor transposon insertion mutant collections.

    更新日期:2017-09-14
  • Preparation of biogenic gas vesicle nanostructures for use as contrast agents for ultrasound and MRI
    Nat. Protoc. (IF 10.032) Pub Date : 2017-09-07
    Anupama Lakshmanan, George J Lu, Arash Farhadi, Suchita P Nety, Martin Kunth, Audrey Lee-Gosselin, David Maresca, Raymond W Bourdeau, Melissa Yin, Judy Yan, Christopher Witte, Dina Malounda, F Stuart Foster, Leif Schröder, Mikhail G Shapiro

    This protocol describes the isolation of gas-filled protein nanostructures, called gas vesicles, their functionalization with moieties for targeting and fluorescence, and how to use them as contrast agents for ultrasound and MRI.

    更新日期:2017-09-07
  • Multiparametric characterization of rare HIV-infected cells using an RNA-flow FISH technique
    Nat. Protoc. (IF 10.032) Pub Date : 2017-09-07
    Amy E Baxter, Julia Niessl, Rémi Fromentin, Jonathan Richard, Filippos Porichis, Marta Massanella, Nathalie Brassard, Nirmin Alsahafi, Jean-Pierre Routy, Andrés Finzi, Nicolas Chomont, Daniel E Kaufmann

    This protocol describes flow cytometric identification of viral translation-competent reservoirs, based on concurrent detection of cellular HIV Gagpol mRNA by in situ RNA hybridization combined with antibody staining for the HIV Gag protein.

    更新日期:2017-09-07
  • Use of a neonatal rat system as a bioincubator to generate adult-like mature cardiomyocytes from human and mouse pluripotent stem cells
    Nat. Protoc. (IF 10.032) Pub Date : 2017-09-07
    Gun-Sik Cho, Emmanouil Tampakakis, Peter Andersen, Chulan Kwon

    This protocol describes how to generate mature adult-like cardiomyocytes by culturing mouse or human PSCs in vitro initially and then transferring to neonatal rats for further cell maturation.

    更新日期:2017-09-07
  • Live-cell confocal microscopy and quantitative 4D image analysis of anchor-cell invasion through the basement membrane in Caenorhabditis elegans
    Nat. Protoc. (IF 10.032) Pub Date : 2017-09-07
    Laura C Kelley, Zheng Wang, Elliott J Hagedorn, Lin Wang, Wanqing Shen, Shijun Lei, Sam A Johnson, David R Sherwood

    This protocol describes how to use multichannel time-lapse confocal imaging of anchor-cell invasion in live Caenorhabditis elegans to monitor cell invasion through basement membranes.

    更新日期:2017-09-07
  • Reconstitution of mouse oogenesis in a dish from pluripotent stem cells
    Nat. Protoc. (IF 10.032) Pub Date : 2017-08-10
    Katsuhiko Hayashi, Orie Hikabe, Yayoi Obata, Yuji Hirao

    This protocol extension describes how to obtain functionally mature oocytes from embryonic or induced pluripotent stem cells. These oocytes can be used to produce live mouse offspring.

    更新日期:2017-09-06
  • The Mini-FLOTAC technique for the diagnosis of helminth and protozoan infections in humans and animals
    Nat. Protoc. (IF 10.032) Pub Date : 2017-08-03
    Giuseppe Cringoli, Maria P Maurelli, Bruno Levecke, Antonio Bosco, Jozef Vercruysse, Jürg Utzinger, Laura Rinaldi

    Here the authors provide an extension of their original FLOTAC protocol, describing the Mini-FLOTAC technique, optimized to perform diagnosis of helminth and protozoan infections in humans and animals where centrifugation may not be practical.

    更新日期:2017-09-06
  • Use of fluorescence-detected sedimentation velocity to study high-affinity protein interactions
    Nat. Protoc. (IF 10.032) Pub Date : 2017-08-03
    Sumit K Chaturvedi, Jia Ma, Huaying Zhao, Peter Schuck

    This protocol describes how to use sedimentation velocity analytical ultracentrifugation in combination with fluorescence optical detection for the analysis of mass, shape, size distribution, and binding constants of interacting proteins.

    更新日期:2017-09-06
  • Mechanical isolation, and measurement of force and myoplasmic free [Ca2+] in fully intact single skeletal muscle fibers
    Nat. Protoc. (IF 10.032) Pub Date : 2017-08-03
    Arthur J Cheng, Håkan Westerblad

    Cheng & Westerblad describe the mechanical dissection of living single fibers from the mouse flexor digitorum brevis muscle. Isolated intact fibers are subsequently used for force and myoplasmic free [Ca2+] measurements.

    更新日期:2017-09-06
  • The cubicon method for concentrating membrane proteins in the cubic mesophase
    Nat. Protoc. (IF 10.032) Pub Date : 2017-08-03
    Pikyee Ma, Dietmar Weichert, Luba A Aleksandrov, Timothy J Jensen, John R Riordan, Xiangyu Liu, Brian K Kobilka, Martin Caffrey

    The lipid cubic phase (in meso) method is used for generating crystals and X-ray structures of integral membrane proteins. This protocol describes the cubicon method for concentrating membrane proteins in the cubic mesophase.

    更新日期:2017-09-06
  • Development of fertile mouse oocytes from mitotic germ cells in vitro
    Nat. Protoc. (IF 10.032) Pub Date : 2017-08-10
    Kanako Morohaku, Yuji Hirao, Yayoi Obata

    This protocol describes how to obtain functionally mature oocytes from primordial germ cells from fetal mouse ovaries in vitro. These oocytes can be used to produce live mouse offspring.

    更新日期:2017-09-06
  • Electron microscopy using the genetically encoded APEX2 tag in cultured mammalian cells
    Nat. Protoc. (IF 10.032) Pub Date : 2017-08-10
    Jeffrey D Martell, Thomas J Deerinck, Stephanie S Lam, Mark H Ellisman, Alice Y Ting

    This protocol describes procedures for using the genetic tag APEX2 to generate contrast for electron microscopy in cultured cells.

    更新日期:2017-09-06
  • Evaluation of telomere length in human cardiac tissues using cardiac quantitative FISH
    Nat. Protoc. (IF 10.032) Pub Date : 2017-08-17
    Maryam Sharifi-Sanjani, Alan K Meeker, Foteini Mourkioti

    This protocol describes how to measure telomere length in archival human cardiac tissues using cardiac quantitative fluorescent in situ hybridization (CQ-FISH) in a cell-type-specific manner.

    更新日期:2017-09-06
  • Meganuclease-assisted generation of stable transgenics in the sea anemone Nematostella vectensis
    Nat. Protoc. (IF 10.032) Pub Date : 2017-08-17
    Eduard Renfer, Ulrich Technau

    The Technau lab provides their protocol for the generation of stably transgenic sea anemones. An expression vector is digested with the meganuclease I-SceI and then microinjected into embryos, where I-SceI mediates stable integration into the genome.

    更新日期:2017-09-06
Some contents have been Reproduced with permission of the American Chemical Society.
Some contents have been Reproduced by permission of The Royal Society of Chemistry.
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