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Nature Protocols

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https://www.readbyqxmd.com/read/29120463/compartmentalized-partnered-replication-for-the-directed-evolution-of-genetic-parts-and-circuits
#1
Zhanar Abil, Jared W Ellefson, Jimmy D Gollihar, Ella Watkins, Andrew D Ellington
Compartmentalized partnered replication (CPR) is an emulsion-based directed evolution method based on a robust and modular phenotype-genotype linkage. In contrast to other in vivo directed evolution approaches, CPR largely mitigates host fitness effects due to a relatively short expression time of the gene of interest. CPR is based on gene circuits in which the selection of a 'partner' function from a library leads to the production of a thermostable polymerase. After library preparation, bacteria produce partner proteins that can potentially lead to enhancement of transcription, translation, gene regulation, and other aspects of cellular metabolism that reinforce thermostable polymerase production...
December 2017: Nature Protocols
https://www.readbyqxmd.com/read/29120462/chromatin-state-discovery-and-genome-annotation-with-chromhmm
#2
Jason Ernst, Manolis Kellis
Noncoding DNA regions have central roles in human biology, evolution, and disease. ChromHMM helps to annotate the noncoding genome using epigenomic information across one or multiple cell types. It combines multiple genome-wide epigenomic maps, and uses combinatorial and spatial mark patterns to infer a complete annotation for each cell type. ChromHMM learns chromatin-state signatures using a multivariate hidden Markov model (HMM) that explicitly models the combinatorial presence or absence of each mark. ChromHMM uses these signatures to generate a genome-wide annotation for each cell type by calculating the most probable state for each genomic segment...
December 2017: Nature Protocols
https://www.readbyqxmd.com/read/29095442/a-high-throughput-in-vivo-screening-method-in-the-mouse-for-identifying-regulators-of-metastatic-colonization
#3
Anneliese O Speak, Agnieszka Swiatkowska, Natasha A Karp, Mark J Arends, David J Adams, Louise van der Weyden
We describe a sensitive, robust, high-throughput method for quantifying the ability of metastatic tumor cells to colonize a secondary organ. Metastasis is the leading cause of death in cancer patients, and successful colonization of the secondary organ is the rate-limiting step in the metastatic process; thus, experimental methods that can be used to interrogate the key factors required for this critical step are of great importance. The experimental metastasis assay we detail here includes tail-vein injection of cancer cells into the mouse and determination of the resulting secondary organ colonization, primarily in the lung, 10 d post dosing...
December 2017: Nature Protocols
https://www.readbyqxmd.com/read/29095441/identification-of-rna-binding-domains-of-rna-binding-proteins-in-cultured-cells-on-a-system-wide-scale-with-rbdmap
#4
Alfredo Castello, Christian K Frese, Bernd Fischer, Aino I Järvelin, Rastislav Horos, Anne-Marie Alleaume, Sophia Foehr, Tomaz Curk, Jeroen Krijgsveld, Matthias W Hentze
This protocol is an extension to: Nat. Protoc. 8, 491-500 (2013); doi:10.1038/nprot.2013.020; published online 14 February 2013RBDmap is a method for identifying, in a proteome-wide manner, the regions of RNA-binding proteins (RBPs) engaged in native interactions with RNA. In brief, cells are irradiated with UV light to induce protein-RNA cross-links. Following stringent denaturing washes, the resulting covalently linked protein-RNA complexes are purified with oligo(dT) magnetic beads. After elution, RBPs are subjected to partial proteolysis, in which the protein regions still bound to the RNA and those released to the supernatant are separated by a second oligo(dT) selection...
December 2017: Nature Protocols
https://www.readbyqxmd.com/read/28079880/nonradioactive-quantification-of-autophagic-protein-degradation-with-l-azidohomoalanine-labeling
#5
Jigang Wang, Jianbin Zhang, Yew Mun Lee, Shukie Ng, Yin Shi, Zi-Chun Hua, Qingsong Lin, Han-Ming Shen
At present, several assays that use radioisotope labeling to quantify the degradation of long-lived proteins have been developed to measure autophagic flux. Here, we describe a nonradioactive pulse-chase protocol using L-azidohomoalanine (AHA) labeling to quantify long-lived protein degradation during autophagy. AHA is used as a surrogate for L-methionine, and, when added to cultured cells grown in methionine-free medium, AHA is incorporated into proteins during de novo protein synthesis. After a chase period to remove short-lived proteins, autophagy is induced by starvation or other stimuli...
December 2017: Nature Protocols
https://www.readbyqxmd.com/read/29072708/preparation-of-glycoconjugates-from-unprotected-carbohydrates-for-protein-binding-studies
#6
Christian T Hjuler, Nicolai N Maolanon, Jørgen Sauer, Jens Stougaard, Mikkel B Thygesen, Knud J Jensen
Glycobiology, in particular the study of carbohydrate-protein interactions and the events that follow, has become an important research focus in recent decades. To study these interactions, many assays require homogeneous glycoconjugates in suitable amounts. Their synthesis is one of the methodological challenges of glycobiology. Here, we describe a versatile, three-stage protocol for the formation of glycoconjugates from unprotected carbohydrates, including those purified from natural sources, as exemplified here by rhizobial Nod factors and exopolysaccharide fragments...
November 2017: Nature Protocols
https://www.readbyqxmd.com/read/29072707/the-assembly-and-use-of-continuous-flow-systems-for-chemical-synthesis
#7
Joshua Britton, Timothy F Jamison
The adoption of and opportunities in continuous flow synthesis ('flow chemistry') have increased significantly over the past several years. Continuous flow systems provide improved reaction safety and accelerated reaction kinetics, and have synthesised several active pharmaceutical ingredients in automated reconfigurable systems. Although continuous flow platforms are commercially available, systems constructed 'in-lab' provide researchers with a flexible, versatile, and cost-effective alternative. Herein, we describe the assembly and use of a modular continuous flow apparatus from readily available and affordable parts in as little as 30 min...
November 2017: Nature Protocols
https://www.readbyqxmd.com/read/29072706/measuring-protein-structural-changes-on-a-proteome-wide-scale-using-limited-proteolysis-coupled-mass-spectrometry
#8
Simone Schopper, Abdullah Kahraman, Pascal Leuenberger, Yuehan Feng, Ilaria Piazza, Oliver Müller, Paul J Boersema, Paola Picotti
Protein structural changes induced by external perturbations or internal cues can profoundly influence protein activity and thus modulate cellular physiology. A number of biophysical approaches are available to probe protein structural changes, but these are not applicable to a whole proteome in a biological extract. Limited proteolysis-coupled mass spectrometry (LiP-MS) is a recently developed proteomics approach that enables the identification of protein structural changes directly in their complex biological context on a proteome-wide scale...
November 2017: Nature Protocols
https://www.readbyqxmd.com/read/29048424/analysis-of-human-cerebrospinal-fluid-monoamines-and-their-cofactors-by-hplc
#9
Marta Batllori, Marta Molero-Luis, Aida Ormazabal, Mercedes Casado, Cristina Sierra, Angels García-Cazorla, Manju Kurian, Simon Pope, Simon J Heales, Rafael Artuch
The presence of monoamines and their cofactors (the pterins and vitamin B6 (pyridoxal phosphate (PLP))) in human cerebrospinal fluid (CSF) can be used as indicators of the biosynthesis and turnover of dopamine and serotonin in the brain. In addition, abnormalities in the CSF levels of these molecules are associated with various neurological diseases, including genetic diseases leading to dopamine and serotonin deficiency. Here, we provide a set of quantitative high-performance liquid-chromatography (HPLC) approaches to determine CSF levels of monoamines and their cofactors...
November 2017: Nature Protocols
https://www.readbyqxmd.com/read/29048423/identification-of-cross-talk-between-sumoylation-and-ubiquitylation-using-a-sequential-peptide-immunopurification-approach
#10
Francis P McManus, Frédéric Lamoliatte, Pierre Thibault
Ubiquitin and ubiquitin-like modifiers (UBLs) such as small ubiquitin-like modifier (SUMO) can act as antagonists to one another by competing to occupy similar residues in the proteome. In addition, SUMO and ubiquitin can be coupled to each other at key lysine residues to form highly branched protein networks. The interplay between these modifications governs important biological processes such as double-strand break repair and meiotic recombination. We recently developed an approach that permits the identification of proteins that are modified by both SUMOylation and ubiquitylation...
November 2017: Nature Protocols
https://www.readbyqxmd.com/read/29048422/cell-derived-matrices-for-studying-cell-proliferation-and-directional-migration-in-a-complex-3d-microenvironment
#11
Riina Kaukonen, Guillaume Jacquemet, Hellyeh Hamidi, Johanna Ivaska
2D surfaces offer simple analysis of cells in culture, yet these often yield different cell morphologies and responses from those observed in vivo. Considerable effort has therefore been expended on the generation of more tissue-like environments for the study of cell behavior in vitro. Purified matrix proteins provide a 3D scaffold that better mimics the in vivo situation; however, these are far removed from the complex tissue composition seen in vivo. Cell-derived matrices (CDMs) offer a more physiologically relevant alternative for studying in vivo-like cell behavior in vitro...
November 2017: Nature Protocols
https://www.readbyqxmd.com/read/29022943/simultaneous-measurement-of-sleep-and-feeding-in-individual-drosophila
#12
Keith R Murphy, Jin Hong Park, Robert Huber, William W Ja
Drosophila is widely used for the dissection of genetic and neuronal mechanisms of behavior. Recently, flies have emerged as a model for investigating the regulation of feeding and sleep. Although typically studied in isolation, increasing evidence points to a fundamental connection between these behaviors. Thus, a system for measuring sleep and feeding simultaneously in a single integrated system is important for interpreting behavioral shifts of either state. Here, we describe the construction and use of the Activity Recording Capillary Feeder or CAFE (ARC), a machine-vision (automated image tracking)-based system for the integrated measurement of sleep and feeding in individual Drosophila...
November 2017: Nature Protocols
https://www.readbyqxmd.com/read/29022942/facile-synthesis-of-gold-nanomaterials-with-unusual-crystal-structures
#13
Zhanxi Fan, Xiao Huang, Ye Chen, Wei Huang, Hua Zhang
Gold (Au) nanomaterials have attracted wide research attention, owing to their high chemical stability, promising catalytic properties, excellent biocompatibility, unique electronic structure and outstanding localized surface plasmon resonance (LSPR) absorption properties; all of which are closely related to their size and shape. Recently, crystal-phase-controlled synthesis of noble metal nanomaterials has emerged as a promising strategy to tune their physicochemical properties. This protocol describes the detailed experimental procedures for the crystal-phase-controlled syntheses of Au nanomaterials with unusual crystal structures under mild conditions...
November 2017: Nature Protocols
https://www.readbyqxmd.com/read/29022941/genome-wide-mapping-of-dnase-i-hypersensitive-sites-in-rare-cell-populations-using-single-cell-dnase-sequencing
#14
James Cooper, Yi Ding, Jiuzhou Song, Keji Zhao
Increased chromatin accessibility is a feature of cell-type-specific cis-regulatory elements; therefore, mapping of DNase I hypersensitive sites (DHSs) enables the detection of active regulatory elements of transcription, including promoters, enhancers, insulators and locus-control regions. Single-cell DNase sequencing (scDNase-seq) is a method of detecting genome-wide DHSs when starting with either single cells or <1,000 cells from primary cell sources. This technique enables genome-wide mapping of hypersensitive sites in a wide range of cell populations that cannot be analyzed using conventional DNase I sequencing because of the requirement for millions of starting cells...
November 2017: Nature Protocols
https://www.readbyqxmd.com/read/28981125/total-chemical-synthesis-of-histones-and-their-analogs-assisted-by-native-chemical-ligation-and-palladium-complexes
#15
Suman Kumar Maity, Muhammad Jbara, Guy Mann, Guy Kamnesky, Ashraf Brik
Chemical synthesis of histones allows precise control of the installation of post-translational modifications via the coupling of derivatized amino acids. Shortcomings of other approaches for obtaining modified histones for epigenetic studies include heterogeneity of the obtained product and difficulties in incorporating multiple modifications on the same histone. In this protocol, unprotected peptide fragments are prepared by Fmoc solid-phase synthesis and coupled in aqueous buffers via native chemical ligation (NCL; in NCL, a peptide bond is formed between a peptide with an N-terminal Cys and another peptide having a C-terminal thioester)...
November 2017: Nature Protocols
https://www.readbyqxmd.com/read/28981124/combining-confocal-and-atomic-force-microscopy-to-quantify-single-virus-binding-to-mammalian-cell-surfaces
#16
Richard Newton, Martin Delguste, Melanie Koehler, Andra C Dumitru, Pawel R Laskowski, Daniel J Müller, David Alsteens
Over the past five years, atomic force microscopy (AFM)-based approaches have evolved into a powerful multiparametric tool set capable of imaging the surfaces of biological samples ranging from single receptors to membranes and tissues. One of these approaches, force-distance curve-based AFM (FD-based AFM), uses a probing tip functionalized with a ligand to image living cells at high-resolution and simultaneously localize and characterize specific ligand-receptor binding events. Analyzing data from FD-based AFM experiments using appropriate probabilistic models allows quantification of the kinetic and thermodynamic parameters that describe the free-energy landscape of the ligand-receptor bond...
November 2017: Nature Protocols
https://www.readbyqxmd.com/read/28981123/a-cerebellar-window-for-intravital-imaging-of-normal-and-disease-states-in-mice
#17
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
The cerebellum is a prominent part of the vertebrate hindbrain that is critically involved in the regulation of important body functions such as movement coordination, maintenance of balance and posture, and motor control. Here, we describe a cerebellar window that provides access to the mouse cerebellum for intravital imaging, thereby allowing for a detailed characterization of the dynamic processes in this region of the brain. First, the skull overlying the cerebellum is removed, and then the window is applied to the region of interest...
November 2017: Nature Protocols
https://www.readbyqxmd.com/read/28981122/base-resolution-stratification-of-cancer-mutations-using-functional-variomics
#18
Song Yi, Ning-Ning Liu, Limei Hu, Hui Wang, Nidhi Sahni
A complete understanding of human cancer variants requires new methods to systematically and efficiently assess the functional effects of genomic mutations at a large scale. Here, we describe a set of tools to rapidly clone and stratify thousands of cancer mutations at base resolution. This protocol provides a massively parallel pipeline to achieve high stringency and throughput. The approach includes high-throughput generation of mutant clones by Gateway, confirmation of variant identity by barcoding and next-generation sequencing, and stratification of cancer variants by multiplexed interaction profiling...
November 2017: Nature Protocols
https://www.readbyqxmd.com/read/28981121/synthesis-and-characterization-of-well-defined-hydrogel-matrices-and-their-application-to-intestinal-stem-cell-and-organoid-culture
#19
Nikolce Gjorevski, Matthias P Lutolf
Growing cells within an extracellular matrix-like 3D gel is required for, or can improve, the growth of many cell types ex vivo. Here, we describe a protocol for the generation of well-defined matrices for the culture of intestinal stem cells (ISCs) and intestinal organoids. These matrices comprise a poly(ethylene glycol) (PEG) hydrogel backbone functionalized with minimal adhesion cues including RGD (Arg-Gly-Asp), which is sufficient for ISC expansion, and laminin-111, which is required for organoid formation...
November 2017: Nature Protocols
https://www.readbyqxmd.com/read/28933779/genetically-encoded-releasable-photo-cross-linking-strategies-for-studying-protein-protein-interactions-in-living-cells
#20
Yi Yang, Haiping Song, Dan He, Shuai Zhang, Shizhong Dai, Xiao Xie, Shixian Lin, Ziyang Hao, Huangtao Zheng, Peng R Chen
Although protein-protein interactions (PPIs) have crucial roles in virtually all cellular processes, the identification of more transient interactions in their biological context remains challenging. Conventional photo-cross-linking strategies can be used to identify transient interactions, but these approaches often suffer from high background due to the cross-linked bait proteins. To solve the problem, we have developed membrane-permeable releasable photo-cross-linkers that allow for prey-bait separation after protein complex isolation and can be installed in proteins of interest (POIs) as unnatural amino acids...
October 2017: Nature Protocols
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