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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/28933779/genetically-encoded-releasable-photo-cross-linking-strategies-for-studying-protein-protein-interactions-in-living-cells
#2
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
https://www.readbyqxmd.com/read/28933778/synthesis-of-a-hycosul-peptide-substrate-library-to-dissect-protease-substrate-specificity
#3
Marcin Poreba, Guy S Salvesen, Marcin Drag
Many biologically and chemically based approaches have been developed to design highly active and selective protease substrates and probes. It is, however, difficult to find substrate sequences that are truly selective for any given protease, as different proteases can demonstrate a great deal of overlap in substrate specificities. In some cases, better enzyme selectivity can be achieved using peptide libraries containing unnatural amino acids such as the hybrid combinatorial substrate library (HyCoSuL), which uses both natural and unnatural amino acids...
October 2017: Nature Protocols
https://www.readbyqxmd.com/read/28933777/generation-and-use-of-a-humanized-bone-marrow-ossicle-niche-for-hematopoietic-xenotransplantation-into-mice
#4
Andreas Reinisch, David Cruz Hernandez, Katharina Schallmoser, Ravindra Majeti
Xenotransplantation is frequently used to study normal and malignant hematopoiesis of human cells. However, conventional mouse xenotransplantation models lack essential human-specific bone-marrow (BM)-microenvironment-derived survival, proliferation, and self-renewal signals for engraftment of normal and malignant blood cells. As a consequence, many human leukemias and other hematologic disorders do not robustly engraft in these conventional models. Here, we describe a complete workflow for the generation of humanized ossicles with an accessible BM microenvironment that faithfully recapitulates normal BM niche morphology and function...
October 2017: Nature Protocols
https://www.readbyqxmd.com/read/28906494/lab-scale-production-of-anhydrous-diazomethane-using-membrane-separation-technology
#5
Doris Dallinger, C Oliver Kappe
Diazomethane is among the most versatile and useful reagents for introducing methyl or methylene groups in organic synthesis. However, because of its explosive nature, its generation and purification by distillation are accompanied by a certain safety risk. This protocol describes how to construct a configurationally simple tube-in-flask reactor for the in situ on-demand generation of anhydrous diazomethane using membrane separation technology and thus avoiding distillation methods. The described reactor can be prepared from commercially available parts within ∼1 h...
October 2017: Nature Protocols
https://www.readbyqxmd.com/read/28906493/rapid-curation-of-gene-disruption-collections-using-knockout-sudoku
#6
Isao A Anzai, Lev Shaket, Oluwakemi Adesina, Michael Baym, Buz Barstow
Knockout Sudoku is a method for the construction of whole-genome knockout collections for a wide range of microorganisms with as little as 3 weeks of dedicated labor and at a cost of ∼$10,000 for a collection for a single organism. The method uses manual 4D combinatorial pooling, next-generation sequencing, and a Bayesian inference algorithm to rapidly process and then accurately annotate the extremely large progenitor transposon insertion mutant collections needed to achieve saturating coverage of complex microbial genomes...
October 2017: Nature Protocols
https://www.readbyqxmd.com/read/28880280/multiparametric-characterization-of-rare-hiv-infected-cells-using-an-rna-flow-fish-technique
#7
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
Efforts to cure HIV are hampered by limited characterization of the cells supporting HIV replication in vivo and inadequate methods for quantifying the latent viral reservoir in individuals receiving antiretroviral therapy (ART). We describe a protocol for flow cytometric identification of viral reservoirs, based on concurrent detection of cellular HIV Gagpol mRNA by in situ RNA hybridization combined with antibody staining for the HIV Gag protein. By simultaneously detecting both HIV RNA and protein, the CD4 T cells harboring translation-competent virus can be identified...
October 2017: Nature Protocols
https://www.readbyqxmd.com/read/28880279/live-cell-confocal-microscopy-and-quantitative-4d-image-analysis-of-anchor-cell-invasion-through-the-basement-membrane-in-caenorhabditis-elegans
#8
Laura C Kelley, Zheng Wang, Elliott J Hagedorn, Lin Wang, Wanqing Shen, Shijun Lei, Sam A Johnson, David R Sherwood
Cell invasion through basement membrane (BM) barriers is crucial in development, leukocyte trafficking and the spread of cancer. The mechanisms that direct invasion, despite their importance in normal and disease states, are poorly understood, largely because of the inability to visualize dynamic cell-BM interactions in vivo. This protocol describes multichannel time-lapse confocal imaging of anchor-cell invasion in live Caenorhabditis elegans. Methods presented include outline-slide preparation and worm growth synchronization (15 min), mounting (20 min), image acquisition (20-180 min), image processing (20 min) and quantitative analysis (variable timing)...
October 2017: Nature Protocols
https://www.readbyqxmd.com/read/28880278/preparation-of-biogenic-gas-vesicle-nanostructures-for-use-as-contrast-agents-for-ultrasound-and-mri
#9
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
Gas vesicles (GVs) are a unique class of gas-filled protein nanostructures that are detectable at subnanomolar concentrations and whose physical properties allow them to serve as highly sensitive imaging agents for ultrasound and MRI. Here we provide a protocol for isolating GVs from native and heterologous host organisms, functionalizing these nanostructures with moieties for targeting and fluorescence, characterizing their biophysical properties and imaging them using ultrasound and MRI. GVs can be isolated from natural cyanobacterial and haloarchaeal host organisms or from Escherichia coli expressing a heterologous GV gene cluster and purified using buoyancy-assisted techniques...
October 2017: Nature Protocols
https://www.readbyqxmd.com/read/28880277/use-of-a-neonatal-rat-system-as-a-bioincubator-to-generate-adult-like-mature-cardiomyocytes-from-human-and-mouse-pluripotent-stem-cells
#10
Gun-Sik Cho, Emmanouil Tampakakis, Peter Andersen, Chulan Kwon
Pluripotent stem cells (PSCs), including induced PSCs, hold great potential for personalized disease modeling, drug testing and cell-based therapeutics. However, cells differentiated from PSCs remain immature in a dish, and thus there are serious caveats to their use in modeling adult-onset diseases such as cardiomyopathies and Alzheimer's disease. By taking advantage of knowledge gained about mammalian development and from bioinformatics analyses, we recently developed a neonatal rat system that enables maturation of PSC-derived cardiomyocytes into cardiomyocytes analogous to those seen in adult animals...
October 2017: Nature Protocols
https://www.readbyqxmd.com/read/28858290/generation-of-high-purity-human-ventral-midbrain-dopaminergic-progenitors-for-in-vitro-maturation-and-intracerebral-transplantation
#11
Sara Nolbrant, Andreas Heuer, Malin Parmar, Agnete Kirkeby
Generation of precisely patterned neural cells from human pluripotent stem cells (hPSCs) is instrumental in developing disease models and stem cell therapies. Here, we provide a detailed 16-d protocol for obtaining high-purity ventral midbrain (VM) dopamine (DA) progenitors for intracerebral transplantation into animal models and for in vitro maturation into neurons. We have successfully transplanted such cells into the rat; however, in principle, the cells can be used for transplantation into any animal model, and the protocol is designed to also be compatible with clinical transplantation into humans...
September 2017: Nature Protocols
https://www.readbyqxmd.com/read/28858289/production-of-tunable-nanomaterials-using-hierarchically-assembled-bacteriophages
#12
Ju Hun Lee, Christopher M Warner, Hyo-Eon Jin, Eftihia Barnes, Aimee R Poda, Edward J Perkins, Seung-Wuk Lee
Large-scale fabrication of precisely defined nanostructures with tunable functions is critical to the exploitation of nanoscience and nanotechnology for production of electronic devices, energy generators, biosensors, and bionanomedicines. Although self-assembly processes have been developed to exploit biological molecules for functional materials, the resulting nanostructures and functions are still very limited, and scalable synthesis is far from being realized. Recently, we have established a bacteriophage-based biomimetic process, called 'self-templating assembly'...
September 2017: Nature Protocols
https://www.readbyqxmd.com/read/28858288/simultaneous-lipid-and-content-mixing-assays-for-in-vitro-reconstitution-studies-of-synaptic-vesicle-fusion
#13
Xiaoxia Liu, Alpay Burak Seven, Junjie Xu, Victoria Esser, Lijing Su, Cong Ma, Josep Rizo
This protocol describes reconstitution assays to study how the neurotransmitter release machinery triggers Ca(2+)-dependent synaptic vesicle fusion. The assays monitor fusion between proteoliposomes containing the synaptic vesicle SNARE synaptobrevin (with or without the Ca(2+) sensor synaptotagmin-1) and proteoliposomes initially containing the plasma membrane SNAREs syntaxin-1 and soluble NSF attachment protein (SNAP)-25. Lipid mixing (from fluorescence de-quenching of Marina-Blue-labeled lipids) and content mixing (from development of fluorescence resonance energy transfer (FRET) between phycoerythrin-biotin (PhycoE-Biotin) and Cy5-streptavidin trapped in the two proteoliposome populations) are measured simultaneously to ensure that true, nonleaky membrane fusion is monitored...
September 2017: Nature Protocols
https://www.readbyqxmd.com/read/28858287/optimized-retroviral-transduction-of-mouse-t-cells-for-in-vivo-assessment-of-gene-function
#14
Makoto Kurachi, Junko Kurachi, Zeyu Chen, John Johnson, Omar Khan, Bertram Bengsch, Erietta Stelekati, John Attanasio, Laura M McLane, Michio Tomura, Satoshi Ueha, E John Wherry
Retroviral (RV) expression of genes of interest (GOIs) is an invaluable tool and has formed the foundation of cellular engineering for adoptive cell therapy in cancer and other diseases. However, monitoring of transduced T cells long term (weeks to months) in vivo remains challenging because of the low frequency and often poor durability of transduced T cells over time when transferred without enrichment. Traditional methods often require additional overnight in vitro culture after transduction. Moreover, in vitro-generated effector CD8(+) T cells enriched by sorting often have reduced viability, making it difficult to monitor the fate of transferred cells in vivo...
September 2017: Nature Protocols
https://www.readbyqxmd.com/read/28837133/construction-of-an-aerolysin-nanopore-in-a-lipid-bilayer-for-single-oligonucleotide-analysis
#15
Chan Cao, Dong-Fang Liao, Jie Yu, He Tian, Yi-Tao Long
Nanopore techniques offer the possibility to study biomolecules at the single-molecule level in a low-cost, label-free and high-throughput manner. By analyzing the level, duration and frequency of ionic current blockades, information regarding the structural conformation, mass, length and concentration of single molecules can be obtained in physiological conditions. Aerolysin monomers assemble into small pores that provide a confined space for effective electrochemical control of a single molecule interacting with the pore, which significantly improves the temporal resolution of this technique...
September 2017: Nature Protocols
https://www.readbyqxmd.com/read/28837132/massively-parallel-and-multiparameter-titration-of-biochemical-assays-with-droplet-microfluidics
#16
Alexandre Baccouche, Shu Okumura, Rémi Sieskind, Elia Henry, Nathanaël Aubert-Kato, Nicolas Bredeche, Jean-François Bartolo, Valérie Taly, Yannick Rondelez, Teruo Fujii, Anthony J Genot
Biochemical systems in which multiple components take part in a given reaction are of increasing interest. Because the interactions between these different components are complex and difficult to predict from basic reaction kinetics, it is important to test for the effect of variations in the concentration for each reagent in a combinatorial manner. For example, in PCR, an increase in the concentration of primers initially increases template amplification, but large amounts of primers result in primer-dimer by-products that inhibit the amplification of the template...
September 2017: Nature Protocols
https://www.readbyqxmd.com/read/28837131/detection-of-nucleic-acid-protein-interactions-in-plant-leaves-using-fluorescence-lifetime-imaging-microscopy
#17
Laurent Camborde, Alain Jauneau, Christian Brière, Laurent Deslandes, Bernard Dumas, Elodie Gaulin
DNA-binding proteins (DNA-BPs) and RNA-binding proteins (RNA-BPs) have critical roles in living cells in all kingdoms of life. Various experimental approaches exist for the study of nucleic acid-protein interactions in vitro and in vivo, but the detection of such interactions at the subcellular level remains challenging. Here we describe how to detect nucleic acid-protein interactions in plant leaves by using a fluorescence resonance energy transfer (FRET) approach coupled to fluorescence lifetime imaging microscopy (FLIM)...
September 2017: Nature Protocols
https://www.readbyqxmd.com/read/28837130/adp-ribose-specific-chromatin-affinity-purification-for-investigating-genome-wide-or-locus-specific-chromatin-adp-ribosylation
#18
Lavinia Bisceglie, Giody Bartolomei, Michael O Hottiger
Protein ADP-ribosylation is a structurally heterogeneous post-translational modification (PTM) that influences the physicochemical and biological properties of the modified protein. ADP-ribosylation of chromatin changes its structural properties, thereby regulating important nuclear functions. A lack of suitable antibodies for chromatin immunoprecipitation (ChIP) has so far prevented a comprehensive analysis of DNA-associated protein ADP-ribosylation. To analyze chromatin ADP-ribosylation, we recently developed a novel ADP-ribose-specific chromatin-affinity purification (ADPr-ChAP) methodology that uses the recently identified ADP-ribose-binding domains RNF146 WWE and Af1521...
September 2017: Nature Protocols
https://www.readbyqxmd.com/read/28817124/directed-differentiation-and-long-term-maintenance-of-epicardial-cells-derived-from-human-pluripotent-stem-cells-under-fully-defined-conditions
#19
Xiaoping Bao, Xiaojun Lian, Tongcheng Qian, Vijesh J Bhute, Tianxiao Han, Sean P Palecek
Here, we describe how to efficiently direct human pluripotent stem cells (hPSCs) differentiation into self-renewing epicardial cells in a completely defined, xeno-free system by temporal modulation of regulators of canonical Wnt signaling. Appropriate differentiation-stage-specific application of Gsk3 inhibitor, Wnt inhibitor, and Gsk3 inhibitor (GiWiGi) is sufficient to produce cells expressing epicardial markers and exhibiting epicardial phenotypes with a high yield and purity from multiple hPSC lines in 16 d...
September 2017: Nature Protocols
https://www.readbyqxmd.com/read/28817123/evaluation-of-telomere-length-in-human-cardiac-tissues-using-cardiac-quantitative-fish
#20
Maryam Sharifi-Sanjani, Alan K Meeker, Foteini Mourkioti
Telomere length has been correlated with various diseases, including cardiovascular disease and cancer. The use of currently available telomere-length measurement techniques is often restricted by the requirement of a large amount of cells (Southern-based techniques) or the lack of information on individual cells or telomeres (PCR-based methods). Although several methods have been used to measure telomere length in tissues as a whole, the assessment of cell-type-specific telomere length provides valuable information on individual cell types...
September 2017: Nature Protocols
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