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Biochemistry

Cell-free Generation of COPII-coated Procollagen I Carriers Authors:  Lin Yuan, Satoshi Baba, Kanika Bajaj and Randy Schekman, date: 11/20/2017, view: 108, Q&A: 0
The aim of this protocol is to generate COPII-coated procollagen I (PC1) carriers in a cell-free reaction. The COPII-coated PC1 carriers were reconstituted from donor membrane, cytosol, purified recombinant COPII proteins, and nucleotides. This protocol describes the preparation of donor membrane and cytosol, the assembly of the reaction, and the ...
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Cell-free Fluorescent Intra-Golgi Retrograde Vesicle Trafficking Assay Authors:  Nathanael P. Cottam and Daniel Ungar, date: 11/20/2017, view: 79, Q&A: 0
Intra-Golgi retrograde vesicle transport is used to traffic and sort resident Golgi enzymes to their appropriate cisternal locations. An assay was established to investigate the molecular details of vesicle targeting in a cell-free system. Stable cell lines were generated in which the trans-Golgi enzyme galactosyltransferase (GalT) was ...
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γ-Secretase Epsilon-cleavage Assay Authors:  Ting-Hai Xu, Yan Yan, Kaleeckal G. Harikumar, Laurence J. Miller, Karsten Melcher and H. Eric Xu, date: 11/20/2017, view: 78, Q&A: 0
γ-Secretase epsilon-cleavage assay is derived from the cell-based Tango assay (Kang et al., 2015), and is a fast and sensitive method to determine the initial cleavage of C99 by γ-secretase. In this protocol, we use HTL cells, which are HEK293 cells with a stably integrated luciferase reporter under the control of the bacterial tetO ...
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Streptavidin Bead Pulldown Assay to Determine Protein Homooligomerization Authors:  Ting-Hai Xu, Yan Yan, Kaleeckal G. Harikumar, Laurence J. Miller, Karsten Melcher and H. Eric Xu, date: 11/20/2017, view: 67, Q&A: 0
Pulldown assay is a conventional method to determine protein-protein interactions in vitro. Expressing a protein of interest with two different tags allows testing whether both versions can be captured via one of the two tags as homooligomeric complex. This protocol is based on streptavidin bead capture of a biotinylated protein and ...
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Bioluminescence Resonance Energy Transfer (BRET) Assay for Determination of Molecular Interactions in Living Cells Authors:  Kaleeckal G. Harikumar, Yan Yan, Ting-Hai Xu, Karsten Melcher, H. Eric Xu and Laurence J. Miller, date: 11/20/2017, view: 114, Q&A: 0
The bioluminescence resonance energy transfer (BRET) assay can be used as an indicator of molecular approximation and/or interaction. A significant resonance energy transfer signal is generated when the acceptor, having the appropriate spectral overlap with the donor emission, is approximated with the donor. In the example provided, proteins ...
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Cancer Biology

An Affinity-directed Protein Missile (AdPROM) System for Targeted Destruction of Endogenous Proteins Authors:  Thomas J Macartney, Gopal P Sapkota and Luke J Fulcher, date: 11/20/2017, view: 66, Q&A: 0
We recently reported an Affinity-directed PROtein Missile (AdPROM) system for the targeted proteolysis of endogenous proteins of interest (POI) (Fulcher et al., 2016 and 2017). AdPROM consists of the Von Hippel Lindau (VHL) protein, a Cullin 2 E3 ligase substrate receptor (Bosu and Kipreos, 2008), conjugated to a high affinity polypeptide ...
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Developmental Biology

Rapid IFM Dissection for Visualizing Fluorescently Tagged Sarcomeric Proteins Authors:  Yu Shu Xiao, Frieder Schöck and Nicanor González-Morales, date: 11/20/2017, view: 80, Q&A: 0
Sarcomeres, the smallest contractile unit of muscles, are arguably the most impressive actomyosin structure. Yet a complete understanding of sarcomere formation and maintenance is missing. The Drosophila indirect flight muscle (IFM) has proven to be a very valuable model to study sarcomeres. Here, we present a protocol for the rapid ...
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Immunology

Instillation of Particulate Suspensions to the Lungs Authors:  Etsushi Kuroda, Yasuo Morimoto and Ken J Ishii, date: 11/20/2017, view: 74, Q&A: 0
Inhaled fine particulates are thought to cause chronic pulmonary inflammation through the deposition of particulates into the lungs. To investigate the effect of fine particulates on the lungs, instillation of suspension of particulates into the lungs is required. This protocol describes direct injection of suspensions of fine particulates into ...
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In vitro Homeostatic Proliferation of Human CD8 T Cells Authors:  Hossam A. Abdelsamed, Caitlin C. Zebley and Ben Youngblood, date: 11/20/2017, view: 53, Q&A: 0
Long-lived T-cell–mediated immunity requires persistence of memory T cells in an antigen-free environment while also maintaining a heightened capacity to recall effector functions. Such antigen-independent homeostatic proliferation is mediated in part by the common gamma-chain cytokines IL-7 and IL-15. To further explore the mechanisms governing ...
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Microbiology

Markerless Gene Editing in the Hyperthermophilic Archaeon Thermococcus kodakarensis Authors:  Alexandra M. Gehring, Travis J. Sanders and Thomas J. Santangelo, date: 11/20/2017, view: 71, Q&A: 0
The advent of single cell genomics and the continued use of metagenomic profiling in diverse environments has exponentially increased the known diversity of life. The recovered and assembled genomes predict physiology, consortium interactions and gene function, but experimental validation of metabolisms and molecular pathways requires more ...
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Organotypic Brain Cultures: A Framework for Studying CNS Infection by Neurotropic Viruses and Screening Antiviral Drugs Authors:  Jeremy Charles Welsch, Claire Lionnet, Christophe Terzian, Branka Horvat, Denis Gerlier and Cyrille Mathieu, date: 11/20/2017, view: 83, Q&A: 0
According to the World Health Organization (WHO), at least 50% of emerging viruses endowed with pathogenicity in humans can infect the Central Nervous System (CNS) with induction of encephalitis and other neurologic diseases (Taylor et al., 2001; Olival and Daszak, 2005). While neurological diseases are progressively documented, the ...
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Quantification of Trypanosoma cruzi in Tissue and Trypanosoma cruzi Killing Assay Authors:  Hisako Kayama, Shoko Kitada and Kiyoshi Takeda, date: 11/20/2017, view: 45, Q&A: 0
Infection with Trypanosoma cruzi causes Chagas disease. The methods provided here allow for the quantification of T. cruzi in the liver, heart, and blood of intraperitoneally-infected mice and analysis of the killing activity of the cells infected with T. cruzi in vitro.
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Infection of Caenorhabditis elegans with Vesicular Stomatitis Virus via Microinjection Authors:  Adam Martin, Emily A. Rex, Takao Ishidate, Rueyling Lin and Don B. Gammon, date: 11/20/2017, view: 53, Q&A: 0
Over the past 15 years, the free-living nematode, Caenorhabditis elegans has become an important model system for exploring eukaryotic innate immunity to bacterial and fungal pathogens. More recently, infection models using either natural or non-natural nematode viruses have also been established in C. elegans. These models offer ...
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Molecular Biology

In vitro Engineered DNA-binding Molecule-mediated Chromatin Immunoprecipitation (in vitro enChIP) Using CRISPR Ribonucleoproteins in Combination with Next-generation Sequencing (in vitro enChIP-Seq) for the Identification of Chromosomal Interactions Authors:  Toshitsugu Fujita and Hodaka Fujii, date: 11/20/2017, view: 53, Q&A: 0
We have developed locus-specific chromatin immunoprecipitation (locus-specific ChIP) technologies consisting of insertional ChIP (iChIP) and engineered DNA-binding molecule-mediated ChIP (enChIP). Locus-specific ChIP is a method to isolate a genomic region of interest from cells while it also identifies what binds to this region using mass ...
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Detection of Membrane Protein Interactions by Cell-based Tango Assays Authors:  Yan Yan, Ting-Hai Xu, Kaleeckal G. Harikumar, Laurence J. Miller, Karsten Melcher and H. Eric Xu, date: 11/20/2017, view: 101, Q&A: 0
The Tango assay is a protein-protein interaction assay, in which a transcription factor (rTA) is fused to a membrane-bound protein via a linker that contains a cleavage site for TEV protease, whereas a soluble interaction partner is fused to TEV protease (Barnea et al., 2008). Association between the two interaction partners leads to an ...
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Neuroscience

Combination of Fluorescent in situ Hybridization (FISH) and Immunofluorescence Imaging for Detection of Cytokine Expression in Microglia/Macrophage Cells Authors:  Maria Fe Lanfranco, David J. Loane, Italo Mocchetti, Mark P. Burns and Sonia Villapol, date: 11/20/2017, view: 106, Q&A: 0
Microglia and macrophage cells are the primary producers of cytokines in response to neuroinflammatory processes. But these cytokines are also produced by other glial cells, endothelial cells, and neurons. It is essential to identify the cells that produce these cytokines to target their different levels of activation. We used dual RNAscope® ...
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Obtaining Multi-electrode Array Recordings from Human Induced Pluripotent Stem Cell–Derived Neurons Authors:  Xiaohong Xu, Carola I. Radulescu, Kagistia Hana Utami and Mahmoud A. Pouladi , date: 11/20/2017, view: 92, Q&A: 0
Neuronal electrical properties are often aberrant in neurological disorders. Human induced pluripotent stem cells (hiPSCs)-derived neurons represent a useful platform for neurological disease modeling, drug discovery and toxicity screening in vitro. Multi-electrode array (MEA) systems offer a non-invasive and label-free platform to record ...
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A Streamlined Method for the Preparation of Gelatin Embedded Brains and Simplified Organization of Sections for Serial Reconstructions Authors:  Andrew W. Liu, Sho Aoki and Jeffery R. Wickens, date: 11/20/2017, view: 143, Q&A: 0
Gelatin embedding of whole brains for sectioning is a critical procedure used in neuroscience to ensure all morphological and spatial details are preserved intact. Here, we describe an inexpensive, reproducible and efficient means to embed post-fixed brains ready for sectioning in gelatin within a week’s time. The sections obtained are ...
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Plant Science

Estimation of Silica Cell Silicification Level in Grass Leaves Using in situ Charring Method Authors:  Santosh Kumar and Rivka Elbaum, date: 11/20/2017, view: 53, Q&A: 0
Silica cells are specialized leaf epidermal cells in grasses with almost the whole cell volume filled with solid silica. In sorghum, silica deposition in silica cells takes place in young, elongating leaves around the mid-length of the leaf. We developed a protocol for estimating the level of silica cell silicification in Sorghum bicolor ...
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Generation and Selection of Transgenic Olive Plants Authors:  Elena Palomo-Ríos, Sergio Cerezo, Jose Ángel Mercado and Fernando Pliego-Alfaro, date: 11/20/2017, view: 55, Q&A: 0
Olive (Olea europaea L.) is one of the most important oil crops in the Mediterranean basin. Biotechnological improvement of this species is hampered by the recalcitrant nature of olive tissue to regenerate in vitro. In previous investigations, our group has developed a reliable Agrobacterium-mediated transformation ...
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Design and Direct Assembly of Synthesized Uracil-containing Non-clonal DNA Fragments into Vectors by USERTM Cloning Authors:  Morten Egevang Jørgensen, Nikolai Wulff, Majse Nafisi, Deyang Xu, Cuiwei Wang, Sophie Konstanze Lambertz, Zeinu Mussa Belew and Hussam Hassan Nour-Eldin, date: 11/20/2017, view: 56, Q&A: 0
This protocol describes how to order and directly assemble uracil-containing non-clonal DNA fragments by uracil excision based cloning (USER cloning). The protocol was generated with the goal of making synthesized non-clonal DNA fragments directly compatible with USERTM cloning. The protocol is highly efficient and would be compatible ...
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