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Nature Protocols

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https://www.readbyqxmd.com/read/28079880/nonradioactive-quantification-of-autophagic-protein-degradation-with-l-azidohomoalanine-labeling
#1
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/28207002/detecting-natural-adaptation-of-the-streptococcus-thermophilus-crispr-cas-systems-in-research-and-classroom-settings
#2
Alexander P Hynes, Marie-Laurence Lemay, Luc Trudel, Hélène Deveau, Michel Frenette, Denise M Tremblay, Sylvain Moineau
CRISPR (clustered regularly interspaced short palindromic repeats)-Cas systems have been adapted into a powerful genome-editing tool. The basis for the flexibility of the tool lies in the adaptive nature of CRISPR-Cas as a bacterial immune system. Here, we describe a protocol to experimentally demonstrate the adaptive nature of this bacterial immune system by challenging the model organism for the study of CRISPR adaptation, Streptococcus thermophilus, with phages in order to detect natural CRISPR immunization...
March 2017: Nature Protocols
https://www.readbyqxmd.com/read/28207001/genome-editing-using-facs-enrichment-of-nuclease-expressing-cells-and-indel-detection-by-amplicon-analysis
#3
Lindsey A Lonowski, Yoshiki Narimatsu, Anjum Riaz, Catherine E Delay, Zhang Yang, Francesco Niola, Katarzyna Duda, Elke A Ober, Henrik Clausen, Hans H Wandall, Steen H Hansen, Eric P Bennett, Morten Frödin
This protocol describes methods for increasing and evaluating the efficiency of genome editing based on the CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats-CRISPR-associated 9) system, transcription activator-like effector nucleases (TALENs) or zinc-finger nucleases (ZFNs). First, Indel Detection by Amplicon Analysis (IDAA) determines the size and frequency of insertions and deletions elicited by nucleases in cells, tissues or embryos through analysis of fluorophore-labeled PCR amplicons covering the nuclease target site by capillary electrophoresis in a sequenator...
March 2017: Nature Protocols
https://www.readbyqxmd.com/read/28207000/spatial-transcriptomic-analysis-of-cryosectioned-tissue-samples-with-geo-seq
#4
Jun Chen, Shengbao Suo, Patrick Pl Tam, Jing-Dong J Han, Guangdun Peng, Naihe Jing
Conventional gene expression studies analyze multiple cells simultaneously or single cells, for which the exact in vivo or in situ position is unknown. Although cellular heterogeneity can be discerned when analyzing single cells, any spatially defined attributes that underpin the heterogeneous nature of the cells cannot be identified. Here, we describe how to use Geo-seq, a method that combines laser capture microdissection (LCM) and single-cell RNA-seq technology. The combination of these two methods enables the elucidation of cellular heterogeneity and spatial variance simultaneously...
March 2017: Nature Protocols
https://www.readbyqxmd.com/read/28182019/a-modular-approach-to-the-design-fabrication-and-characterization-of-muscle-powered-biological-machines
#5
Ritu Raman, Caroline Cvetkovic, Rashid Bashir
Biological machines consisting of cells and biomaterials have the potential to dynamically sense, process, respond, and adapt to environmental signals in real time. As a first step toward the realization of such machines, which will require biological actuators that can generate force and perform mechanical work, we have developed a method of manufacturing modular skeletal muscle actuators that can generate up to 1.7 mN (3.2 kPa) of passive tension force and 300 μN (0.56 kPa) of active tension force in response to external stimulation...
March 2017: Nature Protocols
https://www.readbyqxmd.com/read/28182018/genome-wide-base-resolution-mapping-of-dna-methylation-in-single-cells-using-single-cell-bisulfite-sequencing-scbs-seq
#6
Stephen J Clark, Sébastien A Smallwood, Heather J Lee, Felix Krueger, Wolf Reik, Gavin Kelsey
DNA methylation (DNAme) is an important epigenetic mark in diverse species. Our current understanding of DNAme is based on measurements from bulk cell samples, which obscures intercellular differences and prevents analyses of rare cell types. Thus, the ability to measure DNAme in single cells has the potential to make important contributions to the understanding of several key biological processes, such as embryonic development, disease progression and aging. We have recently reported a method for generating genome-wide DNAme maps from single cells, using single-cell bisulfite sequencing (scBS-seq), allowing the quantitative measurement of DNAme at up to 50% of CpG dinucleotides throughout the mouse genome...
March 2017: Nature Protocols
https://www.readbyqxmd.com/read/28182017/using-connectome-based-predictive-modeling-to-predict-individual-behavior-from-brain-connectivity
#7
Xilin Shen, Emily S Finn, Dustin Scheinost, Monica D Rosenberg, Marvin M Chun, Xenophon Papademetris, R Todd Constable
Neuroimaging is a fast-developing research area in which anatomical and functional images of human brains are collected using techniques such as functional magnetic resonance imaging (fMRI), diffusion tensor imaging (DTI), and electroencephalography (EEG). Technical advances and large-scale data sets have allowed for the development of models capable of predicting individual differences in traits and behavior using brain connectivity measures derived from neuroimaging data. Here, we present connectome-based predictive modeling (CPM), a data-driven protocol for developing predictive models of brain-behavior relationships from connectivity data using cross-validation...
March 2017: Nature Protocols
https://www.readbyqxmd.com/read/28151466/turning-single-molecule-localization-microscopy-into-a-quantitative-bioanalytical-tool
#8
Philip R Nicovich, Dylan M Owen, Katharina Gaus
Single-molecule localization microscopy (SMLM) generates super-resolution images by serially detecting individual fluorescent molecules. The power of SMLM, however, goes beyond images: biologically relevant information can be extracted from the mathematical relationships between the positions of the fluorophores in space and time. Here we review the history of SMLM and how recent progress in methods for spatial point analysis has enabled quantitative measurement of SMLM data, providing insights into biomolecule patterning, clustering and oligomerization in biological systems...
March 2017: Nature Protocols
https://www.readbyqxmd.com/read/28151465/cu-catalyzed-enantioselective-allylic-alkylation-with-organolithium-reagents
#9
Valentín Hornillos, Sureshbabu Guduguntla, Martín Fañanás-Mastral, Manuel Pérez, Pieter H Bos, Alena Rudolph, Syuzanna R Harutyunyan, Ben L Feringa
This protocol describes a method for the catalytic enantioselective synthesis of tertiary and quaternary carbon stereogenic centers, which are widely present in pharmaceutical and natural products. The method is based on the direct reaction between organolithium compounds, which are cheap, readily available and broadly used in chemical synthesis, and allylic electrophiles, using chiral copper catalysts. The methodology involves the asymmetric allylic alkylation (AAA) of allyl bromides, chlorides and ethers with organolithium compounds using catalyst systems based on Cu-Taniaphos and Cu-phosphoramidites...
March 2017: Nature Protocols
https://www.readbyqxmd.com/read/28151464/preparation-of-visible-light-activated-metal-complexes-and-their-use-in-photoredox-nickel-dual-catalysis
#10
Christopher B Kelly, Niki R Patel, David N Primer, Matthieu Jouffroy, John C Tellis, Gary A Molander
Visible-light-activated photoredox catalysts provide synthetic chemists with the unprecedented capability to harness reactive radicals through discrete, single-electron transfer (SET) events. This protocol describes the synthesis of two transition metal complexes, [Ir{dF(CF3)2ppy}2(bpy)]PF6 (1a) and [Ru(bpy)3](PF6)2 (2a), that are activated by visible light. These photoredox catalysts are SET agents that can be used to facilitate transformations ranging from proton-coupled electron-transfer-mediated cyclizations to C-C bond constructions, dehalogenations, and H-atom abstractions...
March 2017: Nature Protocols
https://www.readbyqxmd.com/read/28151463/synthesis-of-a-highly-mg-2-selective-fluorescent-probe-and-its-application-to-quantifying-and-imaging-total-intracellular-magnesium
#11
Azzurra Sargenti, Giovanna Farruggia, Nelsi Zaccheroni, Chiara Marraccini, Massimo Sgarzi, Concettina Cappadone, Emil Malucelli, Alessandra Procopio, Luca Prodi, Marco Lombardo, Stefano Iotti
Magnesium plays a crucial role in many physiological functions and pathological states. Therefore, the evolution of specific and sensitive tools capable of detecting and quantifying this element in cells is a very desirable goal in biological and biomedical research. We developed a Mg(2+)-selective fluorescent dye that can be used to selectively detect and quantify the total magnesium pool in a number of cells that is two orders of magnitude smaller than that required by flame atomic absorption spectroscopy (F-AAS), the reference analytical method for the assessment of cellular total metal content...
March 2017: Nature Protocols
https://www.readbyqxmd.com/read/28125106/strain-library-imaging-protocol-for-high-throughput-automated-single-cell-microscopy-of-large-bacterial-collections-arrayed-on-multiwell-plates
#12
Handuo Shi, Alexandre Colavin, Timothy K Lee, Kerwyn Casey Huang
Single-cell microscopy is a powerful tool for studying gene functions using strain libraries, but it suffers from throughput limitations. Here we describe the Strain Library Imaging Protocol (SLIP), which is a high-throughput, automated microscopy workflow for large strain collections that requires minimal user involvement. SLIP involves transferring arrayed bacterial cultures from multiwell plates onto large agar pads using inexpensive replicator pins and automatically imaging the resulting single cells. The acquired images are subsequently reviewed and analyzed by custom MATLAB scripts that segment single-cell contours and extract quantitative metrics...
February 2017: Nature Protocols
https://www.readbyqxmd.com/read/28125105/conditional-reprogramming-and-long-term-expansion-of-normal-and-tumor-cells-from-human-biospecimens
#13
Xuefeng Liu, Ewa Krawczyk, Frank A Suprynowicz, Nancy Palechor-Ceron, Hang Yuan, Aleksandra Dakic, Vera Simic, Yun-Ling Zheng, Praathibha Sripadhan, Chen Chen, Jie Lu, Tung-Wei Hou, Sujata Choudhury, Bhaskar Kallakury, Dean Tang, Thomas Darling, Rajesh Thangapazham, Olga Timofeeva, Anatoly Dritschilo, Scott H Randell, Christopher Albanese, Seema Agarwal, Richard Schlegel
Historically, it has been difficult to propagate cells in vitro that are derived directly from human tumors or healthy tissue. However, in vitro preclinical models are essential tools for both the study of basic cancer biology and the promotion of translational research, including drug discovery and drug target identification. This protocol describes conditional reprogramming (CR), which involves coculture of irradiated mouse fibroblast feeder cells with normal and tumor human epithelial cells in the presence of a Rho kinase inhibitor (Y-27632)...
February 2017: Nature Protocols
https://www.readbyqxmd.com/read/28125104/modeling-and-docking-of-antibody-structures-with-rosetta
#14
Brian D Weitzner, Jeliazko R Jeliazkov, Sergey Lyskov, Nicholas Marze, Daisuke Kuroda, Rahel Frick, Jared Adolf-Bryfogle, Naireeta Biswas, Roland L Dunbrack, Jeffrey J Gray
We describe Rosetta-based computational protocols for predicting the 3D structure of an antibody from sequence (RosettaAntibody) and then docking the antibody to protein antigens (SnugDock). Antibody modeling leverages canonical loop conformations to graft large segments from experimentally determined structures, as well as offering (i) energetic calculations to minimize loops, (ii) docking methodology to refine the VL-VH relative orientation and (iii) de novo prediction of the elusive complementarity determining region (CDR) H3 loop...
February 2017: Nature Protocols
https://www.readbyqxmd.com/read/28125103/n-formylation-and-n-methylation-of-amines-using-metal-free-n-heterocyclic-carbene-catalysts-and-co2-as-carbon-source
#15
Felix D Bobbink, Shoubhik Das, Paul J Dyson
N-formylation and N-methylation of amines are important reactions that are used to produce a wide range of key intermediates and compounds. This protocol describes the environmentally benign N-formylation and N-methylation of primary and secondary amines using carbon dioxide (CO2) as the carbon source, hydrosilanes as reductants and N-heterocyclic carbenes (NHCs) as catalysts. Using CO2 as a reagent has the advantage of low cost and negligible toxicity. However, the catalyst is air-sensitive and must be generated fresh before use; consequently, the techniques used to prepare and manipulate the catalyst are described...
February 2017: Nature Protocols
https://www.readbyqxmd.com/read/28125102/use-of-the-supported-membrane-tube-assay-system-for-real-time-analysis-of-membrane-fission-reactions
#16
Srishti Dar, Sukrut C Kamerkar, Thomas J Pucadyil
The process of membrane fission is fundamental to diverse cellular processes such as nutrient uptake, synaptic transmission and organelle biogenesis, and it involves the localized application of curvature stress to a tubular membrane intermediate, forcing it to undergo scission. Alternative techniques for creating such substrates necessitate the use of micromanipulators or sophisticated optical traps and require a high level of technical expertise. We present a facile method to generate an array of membrane tubes supported on a passivated glass coverslip, which we refer to as supported membrane tubes (SMrTs)...
February 2017: Nature Protocols
https://www.readbyqxmd.com/read/28102837/precise-and-efficient-scarless-genome-editing-in-stem-cells-using-correct
#17
Dylan Kwart, Dominik Paquet, Shaun Teo, Marc Tessier-Lavigne
CRISPR/Cas9 is a promising tool for genome-editing DNA in cells with single-base-pair precision, which allows novel in vitro models of human disease to be generated-e.g., in pluripotent stem cells. However, the accuracy of intended sequence changes can be severely diminished by CRISPR/Cas9's propensity to re-edit previously modified loci, causing unwanted mutations (indels) alongside intended changes. Here we describe a genome-editing framework termed consecutive re-guide or re-Cas steps to erase CRISPR/Cas-blocked targets (CORRECT), which, by exploiting the use of highly efficacious CRISPR/Cas-blocking mutations in two rounds of genome editing, enables accurate, efficient and scarless introduction of specific base changes-for example, in human induced pluripotent (iPS) stem cells...
February 2017: Nature Protocols
https://www.readbyqxmd.com/read/28102836/preparation-characterization-and-in-vitro-dosimetry-of-dispersed-engineered-nanomaterials
#18
Glen M DeLoid, Joel M Cohen, Georgios Pyrgiotakis, Philip Demokritou
Evidence continues to grow of the importance of in vitro and in vivo dosimetry in the hazard assessment and ranking of engineered nanomaterials (ENMs). Accurate dose metrics are particularly important for in vitro cellular screening to assess the potential health risks or bioactivity of ENMs. To ensure meaningful and reproducible quantification of in vitro dose, with consistent measurement and reporting between laboratories, it is necessary to adopt standardized and integrated methodologies for (i) generation of stable ENM suspensions in cell culture media; (ii) colloidal characterization of suspended ENMs, particularly of properties that determine particle kinetics in an in vitro system (size distribution and formed agglomerate effective density); and (iii) robust numerical fate and transport modeling for accurate determination of the ENM dose delivered to cells over the course of the in vitro exposure...
February 2017: Nature Protocols
https://www.readbyqxmd.com/read/28102835/spinal-cord-regeneration-in-xenopus-laevis
#19
Gabriela Edwards-Faret, Rosana Muñoz, Emilio E Méndez-Olivos, Dasfne Lee-Liu, Victor S Tapia, Juan Larraín
Here we present a protocol for the husbandry of Xenopus laevis tadpoles and froglets, and procedures to study spinal cord regeneration. This includes methods to induce spinal cord injury (SCI); DNA and morpholino electroporation for genetic studies; in vivo imaging for cell analysis; a swimming test to measure functional recovery; and a convenient model for screening for new compounds that promote neural regeneration. These protocols establish X. laevis as a unique model organism for understanding spinal cord regeneration by comparing regenerative and nonregenerative stages...
February 2017: Nature Protocols
https://www.readbyqxmd.com/read/28079879/the-cluspro-web-server-for-protein-protein-docking
#20
Dima Kozakov, David R Hall, Bing Xia, Kathryn A Porter, Dzmitry Padhorny, Christine Yueh, Dmitri Beglov, Sandor Vajda
The ClusPro server (https://cluspro.org) is a widely used tool for protein-protein docking. The server provides a simple home page for basic use, requiring only two files in Protein Data Bank (PDB) format. However, ClusPro also offers a number of advanced options to modify the search; these include the removal of unstructured protein regions, application of attraction or repulsion, accounting for pairwise distance restraints, construction of homo-multimers, consideration of small-angle X-ray scattering (SAXS) data, and location of heparin-binding sites...
February 2017: Nature Protocols
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