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Biochemistry

Measurement of RNA-induced PKR Activation in vitro Author:  Kobe C. Yuen, date: 03/20/2017, view: 1655, Q&A: 0
Protein kinase R (PKR) is one of the key RNA-activated sensors for innate immunity. PKR is activated by pathogenic or aberrant RNAs such as short double-stranded RNAs or those with imperfect secondary structures, as well as a reduction in the amount and number of RNA modifications. Activation of PKR may be an underlying mechanism for the ...
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Cancer Biology

Polysome Analysis Authors:  Dipak Kumar Poria and Partho Sarothi Ray, date: 03/20/2017, view: 1927, Q&A: 1
Polysome analysis is a method to separate mRNAs from a cell into actively translating and non-translating fractions depending on their association with polysomes. By this protocol, cell lysates are fractionated by sucrose density gradient ultracentrifugation. Free mRNA fraction and various ribosomal fractions, such as 40S, 60S, monosomes and ...
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RNA-protein UV-crosslinking Assay Authors:  Dipak Kumar Poria and Partho Sarothi Ray, date: 03/20/2017, view: 2430, Q&A: 2
RNA-protein interactions play a crucial role in every aspect of RNA metabolism, and also plays a major role in post-transcriptional gene regulation. RNA-binding proteins have been implicated in viral gene expression (Ray and Das, 2002) and microRNA-mediated gene regulation (Poria et al., 2016). Here we have described the protocol which ...
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3D Stroma Invasion Assay Authors:  Yvette May Coulson-Thomas and Vivien Jane Coulson-Thomas, date: 03/20/2017, view: 1494, Q&A: 0
We have developed a 3D co-culture system composed of fibroblasts and colorectal cancer cells that enables us to study the desmoplastic reaction. This method also enables us to study the influence of the desmoplastic reaction on the migration of colorectal cancer cells through the surrounding stroma. This protocol has been previously published ...
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Developmental Biology

Measuring Caenorhabditis elegans Sleep during the Transition to Adulthood Using a Microfluidics-based System Authors:  Huiyan Huang, Komudi Singh and Anne C. Hart, date: 03/20/2017, view: 1870, Q&A: 0
C. elegans sleep during development is regulated by genes and cellular mechanisms that are conserved across the animal kingdom (Singh et al., 2014; Trojanowski and Raizen, 2016). C. elegans developmental sleep is usually assessed during the transition to adulthood, a 2.6 h time interval called lethargus (Raizen et al ...
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Immunology

Mouse Model of Reversible Intestinal Inflammation Authors:  Cheong KC Kwong Chung, Jennifer Brasseit, Esther Althaus-Steiner, Silvia Rihs and Christoph Mueller, date: 03/20/2017, view: 1915, Q&A: 0
Current therapies to treat inflammatory bowel disease by dampening excessive inflammatory immune responses have had limited success (Reinisch et al., 2011; Rutgeerts et al., 2005; Sandborn et al., 2012). To develop new therapeutic interventions, there is a need for better understanding of the mechanisms that are ...
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Differential Salt Fractionation of Nuclei to Analyze Chromatin-associated Proteins from Cultured Mammalian Cells Authors:  Christin Herrmann, Daphne C. Avgousti and Matthew D. Weitzman, date: 03/20/2017, view: 2809, Q&A: 0
Nucleosomes are the core units of cellular chromatin and are comprised of 147 base pairs (bp) of DNA wrapped around an octamer of histone proteins. Proteins such as chromatin remodelers, transcription factors, and DNA repair proteins interact dynamically with chromatin to regulate access to DNA, control gene transcription, and maintain genome ...
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Adoptive Transfer of Lung Antigen Presenting Cells Authors:  Xiaofeng Zhou and Bethany B Moore, date: 03/20/2017, view: 1910, Q&A: 0
Our protocol describes adoptive transfer of antigen presenting cells (APCs) isolated from the lungs by enzymatic digestion and magnetic enrichment. This protocol can be used to study APC functions and trafficking.
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Measurement of Dipeptidylpeptidase Activity in vitro and in vivo Authors:  Rosa Barreira da Silva, Molly A. Ingersoll and Matthew L. Albert, date: 03/20/2017, view: 1517, Q&A: 0
Dipeptidylpeptidases (DPPs) are serine proteases, which cleave small proteins and peptides possessing a proline or an alanine in the second position of their N-terminus. Among the members of this family, dipeptidylpeptidase 4 (DPP4) is constitutively expressed in the extracellular space. DPP4 is found at the surface of many hematopoietic and ...
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Mouse CD8+ T Cell Migration in vitro and CXCR3 Internalization Assays Authors:  Rosa Barreira da Silva and Matthew L. Albert, date: 03/20/2017, view: 2165, Q&A: 0
Chemokines are molecules that regulate the positioning of cells during homeostasis and inflammation. CXCL10 is an interferon-induced chemokine that attracts cells that express the chemokine receptor CXCR3 on their surface. CXCL10 expression is often induced upon inflammation and guides lymphocytes, such as T and NK cells, into the injured tissues. ...
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MHC Class II Tetramer Labeling of Human Primary CD4+ T Cells from HIV Infected Patients Authors:  Moran Galperin, Daniela Benati, Mathieu Claireaux, Madhura Mukhopadhyay and Lisa A. Chakrabarti, date: 03/20/2017, view: 1858, Q&A: 0
Major Histocompatibility Complex (MHC) tetramers have been used for two decades to detect, isolate and characterize T cells specific for various pathogens and tumor antigens. In the context of Human Immunodeficiency Virus (HIV) infection, antigen-specific CD8+ T cells have been extensively studied ex vivo, as they can be ...
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Microbiology

Isolation of Ribosomal Particles from the Unicellular Cyanobacterium Synechocystis sp. PCC 6803 Authors:  Carla V. Galmozzi and M. Isabel Muro-Pastor , date: 03/20/2017, view: 1535, Q&A: 0
Isolation of ribosomal particles is an essential step in the study of ribosomal components as well as in the analysis of trans-acting factors that interact with the ribosome to regulate protein synthesis and modulate the expression profile of the cell in response to different environmental conditions. In this protocol, we describe a ...
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Determination of the Predatory Capability of Bdellovibrio bacteriovorus HD100 Authors:  Cristina Herencias, M. Auxiliadora Prieto and Virginia Martínez, date: 03/20/2017, view: 1951, Q&A: 0
Bdellovibrio bacteriovorus HD100 is an obligate predator that preys upon a wide variety of Gram negative bacteria. The biphasic growth cycle of Bdellovibrio includes a free-swimming attack phase and an intraperiplasmic growth phase, where the predator replicates its DNA and grows using the prey as a source of nutrients, finally ...
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In vitro Assay to Assess Efficacy of Potential Antiviral Compounds against Enterovirus D68 Authors:  Liang Sun, Leen Delang, Carmen Mirabelli and Johan Neyts, date: 03/20/2017, view: 1658, Q&A: 0
In 2014 enterovirus D68 (EV-D68) caused the largest outbreak in the United States since the discovery of the virus. Distinct from before, the 2014 infections were associated with more severe respiratory disease and occasional neurological complications. So far, there are no available vaccines or antivirals for the prophylaxis or treatment of ...
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Chase Assay of Protein Stability in Haloferax volcanii Authors:  Xian Fu and Julie A. Maupin-Furlow, date: 03/20/2017, view: 1431, Q&A: 0
Highly regulated and targeted protein degradation plays a fundamental role in almost all cellular processes. Determination of the protein half-life by the chase assay serves as a powerful and popular strategy to compare the protein stability and study proteolysis pathways in cells. Here, we describe a chase assay in Haloferax volcanii, a ...
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Assays for the Detection of Rubber Oxygenase Activities Authors:  Wolf Röther, Jakob Birke and Dieter Jendrossek, date: 03/20/2017, view: 1643, Q&A: 0
Microbial biodegradation of rubber relies on extracellular rubber oxygenases that catalyze the oxidative cleavage of the double bond of the polyisoprene backbone into oligo-isoprenoids. This protocol describes the determination of rubber oxygenase activities by an online measurement of molecular oxygen consumption via a non-invasive ...
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Wheat Root-dip Inoculation with Fusarium graminearum and Assessment of Root Rot Disease Severity Authors:  Qing Wang and Sven Gottwald, date: 03/20/2017, view: 2014, Q&A: 0
Fusarium graminearum is one of the most common and potent fungal pathogens of wheat (Triticum aestivum) and other cereals, known for causing devastating yield losses and mycotoxin contaminations of food and feed. The pathogen is mainly considered as a paradigm for the floral disease Fusarium head blight, while its ability to ...
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Extraction, Purification and Quantification of Diffusible Signal Factor Family Quorum-sensing Signal Molecules in Xanthomonas oryzae pv. oryzae Authors:  Lian Zhou, Xing-Yu Wang, Wei Zhang, Shuang Sun and Ya-Wen He, date: 03/20/2017, view: 1999, Q&A: 0
Bacteria use quorum-sensing (QS) systems to monitor and regulate their population density. Bacterial QS involves small molecules that act as signals for bacterial communication. Many Gram-negative bacterial pathogens use a class of widely conserved molecules, called diffusible signal factor (DSF) family QS signals. The measurement of DSF family ...
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Determination of Adeno-associated Virus Rep DNA Binding Using Fluorescence Anisotropy Authors:  Francisco Zarate-Perez, Vishaka Santosh, Martino Bardelli, Leticia Agundez, R. Michael Linden, Els Henckaerts and Carlos R. Escalante, date: 03/20/2017, view: 1510, Q&A: 0
Quantitative measurement of proteins binding to DNA is a requisite to fully characterize the structural determinants of complex formation necessary to understand the DNA transactions that regulate cellular processes. Here we describe a detailed protocol to measure binding affinity of the adeno-associated virus (AAV) Rep68 protein for the ...
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Molecular Biology

In silico Analysis and Site-directed Mutagenesis of Promoters Authors:  Salah Boudjadi and Jean-Francois Beaulieu, date: 03/20/2017, view: 1840, Q&A: 0
In normal as in cancerous cells, gene expression is tightly regulated by transcription factors, which are responsible for up- or down-regulation of thousands of targets involved in different cell processes. Transcription factors can directly regulate the expression of genes by binding to specific DNA sequences known as response elements. ...
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Plant Science

Pathogenicity Assay of Verticillium nonalfalfae on Hop Plants Authors:  Marko Flajšman, Sebastjan Radišek and Branka Javornik, date: 03/20/2017, view: 1785, Q&A: 0
Verticillium nonalfalfae is a soil-borne plant pathogen that infects its hosts through roots. It spreads in the plant’s xylem and causes wilt disease symptoms by secreting different virulence factors. Hop (Humulus lupulus) is a primary host of V. nonalfalfae, so it is used as a model plant for studying this ...
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Xylem Sap Extraction Method from Hop Plants Authors:  Marko Flajšman, Stanislav Mandelc, Sebastjan Radišek and Branka Javornik, date: 03/20/2017, view: 2115, Q&A: 0
Verticillium wilt is one of the most important diseases on hop that significantly influence continuation of production on affected areas. It is caused by the soil borne vascular pathogen Verticillium nonalfalfae, which infects plants through the roots and then advances through the vascular (xylem) system. During infection, V. nonalfalfae ...
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Extraction and Analysis of Carotenoids from Escherichia coli in Color Complementation Assays Authors:  Andreas Blatt and Martin Lohr, date: 03/20/2017, view: 2043, Q&A: 0
A common method to investigate the function of genes putatively involved in carotenoid biosynthesis is the so called color complementation assay in Escherichia coli (see, e.g., Cunningham and Gantt, 2007). In this assay, the gene under investigation is expressed in E. coli strains genetically engineered to synthesize ...
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Ribosomal RNA N-glycosylase Activity Assay of Ribosome-inactivating Proteins Authors:  Rosario Iglesias, Lucía Citores and José M. Ferreras, date: 03/20/2017, view: 2394, Q&A: 0
Ribosome-inactivating proteins (RIPs) are enzymes that irreversibly inactivate ribosomes as a consequence of their N-glycosylase (EC 3.2.2.22) activity. The enzyme cleaves the N-glycosidic bond between the adenine No. 4324 from the 28S rRNA and its ribose in rat ribosomes (or the equivalent adenine in sensitive ribosomes from other organisms). ...
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Rubisco Extraction and Purification from Diatoms Authors:  Jodi N. Young, Ana M. C. Heureux, Rosalind E. M. Rickaby, François M. M. Morel, Spencer M. Whitney and Robert E. Sharwood, date: 03/20/2017, view: 1954, Q&A: 0
This protocol describes a method to extract ribulose-1,5-bisphosphate carboxylase oxygenase (Rubisco) from diatoms (Bacillariophyta) to determine catalytic performance. This protocol has been adapted from use in cyanobacteria and higher plants (Andrews, 1988; Whitney and Sharwood, 2007). First part (steps A1-A3) of the extraction provides ...
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