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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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).
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
Some contents have been Reproduced by permission of The Royal Society of Chemistry.
- Acc. Chem. Res.
- ACS Appl. Mater. Interfaces
- ACS Biomater. Sci. Eng.
- ACS Catal.
- ACS Cent. Sci.
- ACS Chem. Biol.
- ACS Chem. Neurosci.
- ACS Comb. Sci.
- ACS Earth Space Chem.
- ACS Energy Lett.
- ACS Infect. Dis.
- ACS Macro Lett.
- ACS Med. Chem. Lett.
- ACS Nano
- ACS Omega
- ACS Photonics
- ACS Sens.
- ACS Sustainable Chem. Eng.
- ACS Synth. Biol.
- Acta Biomater.
- Acta Mater.
- Adv. Colloid Interface Sci.
- Adv. Electron. Mater.
- Adv. Energy Mater.
- Adv. Funct. Mater.
- Adv. Healthcare Mater.
- Adv. Mater.
- Adv. Mater. Interfaces
- Adv. Opt. Mater.
- Adv. Sci.
- Adv. Synth. Catal.
- AlChE J.
- Anal. Bioanal. Chem.
- Anal. Chem.
- Anal. Chim. Acta
- Anal. Methods
- Angew. Chem. Int. Ed.
- Annu. Rev. Anal. Chem.
- Annu. Rev. Biochem.
- Annu. Rev. Food Sci. Technol.
- Annu. Rev. Mater. Res.
- Annu. Rev. Phys. Chem.
- Appl. Catal. A Gen.
- Appl. Catal. B Environ.
- Appl. Clay. Sci.
- Appl. Energy
- Aquat. Toxicol.
- Arab. J. Chem.
- Asian J. Org. Chem.
- Atmos. Environ.
- Carbohydr. Polym.
- Catal. Commun.
- Catal. Sci. Technol.
- Catal. Today
- Cell Chem. Bio.
- Cem. Concr. Res.
- Ceram. Int.
- Chem. Asian J.
- Chem. Bio. Drug Des.
- Chem. Biol. Interact.
- Chem. Commun.
- Chem. Educ. Res. Pract.
- Chem. Eng. J.
- Chem. Eng. Sci.
- Chem. Eur. J.
- Chem. Mater.
- Chem. Phys.
- Chem. Phys. Lett.
- Chem. Phys. Lipids
- Chem. Rev.
- Chem. Sci.
- Chem. Soc. Rev.
- Combust. Flame
- Compos. Part A Appl. Sci. Manuf.
- Compos. Sci. Technol.
- Compr. Rev. Food Sci. Food Saf.
- Comput. Chem. Eng.
- Constr. Build. Mater.
- Coordin. Chem. Rev.
- Corros. Sci.
- Crit. Rev. Food Sci. Nutr.
- Crit. Rev. Solid State Mater. Sci.
- Cryst. Growth Des.
- Curr. Opin. Chem. Eng.
- Curr. Opin. Colloid Interface Sci.
- Curr. Opin. Environ. Sustain
- Curr. Opin. Solid State Mater. Sci.
- Ecotox. Environ. Safe.
- Electrochem. Commun.
- Electrochim. Acta
- Energy Environ. Sci.
- Energy Fuels
- Environ. Impact Assess. Rev.
- Environ. Int.
- Environ. Model. Softw.
- Environ. Pollut.
- Environ. Res.
- Environ. Sci. Policy
- Environ. Sci. Technol.
- Environ. Sci. Technol. Lett.
- Environ. Sci.: Nano
- Environ. Sci.: Processes Impacts
- Environ. Sci.: Water Res. Technol.
- Eur. J. Inorg. Chem.
- Eur. J. Med. Chem.
- Eur. J. Org. Chem.
- Eur. Polym. J.
- J. Acad. Nutr. Diet.
- J. Agric. Food Chem.
- J. Alloys Compd.
- J. Am. Ceram. Soc.
- J. Am. Chem. Soc.
- J. Am. Soc. Mass Spectrom.
- J. Anal. Appl. Pyrol.
- J. Anal. At. Spectrom.
- J. Antibiot.
- J. Catal.
- J. Chem. Educ.
- J. Chem. Eng. Data
- J. Chem. Inf. Model.
- J. Chem. Phys.
- J. Chem. Theory Comput.
- J. Chromatogr. A
- J. Chromatogr. B
- J. Clean. Prod.
- J. CO2 UTIL.
- J. Colloid Interface Sci.
- J. Comput. Chem.
- J. Cryst. Growth
- J. Dairy Sci.
- J. Electroanal. Chem.
- J. Electrochem. Soc.
- J. Environ. Manage.
- J. Eur. Ceram. Soc.
- J. Fluorine Chem.
- J. Food Drug Anal.
- J. Food Eng.
- J. Food Sci.
- J. Funct. Foods
- J. Hazard. Mater.
- J. Hydrol.
- J. Ind. Eng. Chem.
- J. Inorg. Biochem.
- J. Magn. Magn. Mater.
- J. Mater. Chem. A
- J. Mater. Chem. B
- J. Mater. Chem. C
- J. Mater. Process. Tech.
- J. Mech. Behav. Biomed. Mater.
- J. Med. Chem.
- J. Membr. Sci.
- J. Mol. Catal. A Chem.
- J. Mol. Liq.
- J. Nat. Gas Sci. Eng.
- J. Nat. Prod.
- J. Nucl. Mater.
- J. Org. Chem.
- J. Photochem. Photobiol. C Photochem. Rev.
- J. Phys. Chem. A
- J. Phys. Chem. B
- J. Phys. Chem. C
- J. Phys. Chem. Lett.
- J. Porphyr. Phthalocyanines
- J. Power Sources
- J. Solid State Chem.
- J. Taiwan Inst. Chem. E.
- Macromol. Rapid Commun.
- Mass Spectrom. Rev.
- Mater. Chem. Front.
- Mater. Des.
- Mater. Horiz.
- Mater. Lett.
- Mater. Sci. Eng. A
- Mater. Sci. Eng. R Rep.
- Mater. Today
- Meat Sci.
- Med. Chem. Commun.
- Microchem. J.
- Microchim. Acta
- Micropor. Mesopor. Mater.
- Mol. Biosyst.
- Mol. Cancer Ther.
- Mol. Catal.
- Mol. Nutr. Food Res.
- Mol. Pharmaceutics
- Mol. Syst. Des. Eng.
- Nano Energy
- Nano Lett.
- Nano Res.
- Nano Today
- Nano-Micro Lett.
- Nanoscale Horiz.
- Nat. Catal.
- Nat. Chem.
- Nat. Chem. Biol.
- Nat. Commun.
- Nat. Energy
- Nat. Mater.
- Nat. Med.
- Nat. Methods
- Nat. Nanotech.
- Nat. Photon.
- Nat. Prod. Rep.
- Nat. Protoc.
- Nat. Rev. Chem.
- Nat. Rev. Drug. Disc.
- Nat. Rev. Mater.
- Neurochem. Int.
- New J. Chem.
- NPG Asia Mater.
- npj 2D Mater. Appl.
- npj Comput. Mater.
- npj Flex. Electron.
- npj Mater. Degrad.
- npj Sci. Food
- Pharmacol. Rev.
- Pharmacol. Therapeut.
- Photochem. Photobiol. Sci.
- Phys. Chem. Chem. Phys.
- Phys. Life Rev.
- PLOS ONE
- Polym. Chem.
- Polym. Degrad. Stabil.
- Polym. J.
- Polym. Rev.
- Powder Technol.
- Proc. Combust. Inst.
- Prog. Cryst. Growth Ch. Mater.
- Prog. Energy Combust. Sci.
- Prog. Mater. Sci.
- Prog. Photovoltaics
- Prog. Polym. Sci.
- Prog. Solid State Chem.