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Raquel R Martins, Andrew W McCracken, Mirre J P Simons, Catarina M Henriques, Michael Rera
The Smurf Assay (SA) was initially developed in the model organism Drosophila melanogaster where a dramatic increase of intestinal permeability has been shown to occur during aging (Rera et al. , 2011). We have since validated the protocol in multiple other model organisms (Dambroise et al. , 2016) and have utilized the assay to further our understanding of aging (Tricoire and Rera, 2015; Rera et al. , 2018). The SA has now also been used by other labs to assess intestinal barrier permeability (Clark et al...
February 5, 2018: Bio-protocol
Sviatlana Shashkova, Adam Jm Wollman, Stefan Hohmann, Mark C Leake
Single-molecule fluorescence microscopy enables unrivaled sub-cellular quantitation of genomically encoded fusions of native proteins with fluorescent protein reporters. Fluorescent proteins must undergo in vivo maturation after expression before they become photoactive. Maturation effects must be quantified during single-molecule analysis. Here we present a method to characterise maturation of GFP and mCherry genetic protein fusions in budding yeast Saccharomyces cerevisiae.
January 20, 2018: Bio-protocol
Thotegowdanapalya C Mohan, Alexandra M E Jones
To determine boron quantity in soil, water and biological samples, several protocols are available. Colorimetric assays are the simplest and cheapest methods which can be used to determine boron concentration. However, published protocols do not give straightforward guidance for beginners to adopt these protocols for routine use in the laboratory. Based on a previously published available procedure, we present a detailed and modified version of a curcumin based colorimetric protocol to determine boron concentration extracted from any sample...
January 20, 2018: Bio-protocol
Anthony J Snyder, Pranav Danthi
The mammalian orthoreovirus (reovirus) outer capsid undergoes a series of conformational changes prior to or during viral entry. These transitions are necessary for delivering the genome-containing core across host cell membranes. This protocol describes an in vitro assay for monitoring the transition into a membrane penetration-active form (i.e., ISVP*).
January 20, 2018: Bio-protocol
Lanxin Li, S F Gabriel Krens, Matyáš Fendrych, Jiří Friml
The rapid auxin-triggered growth of the Arabidopsis hypocotyls involves the nuclear TIR1/AFB-Aux/IAA signaling and is accompanied by acidification of the apoplast and cell walls (Fendrych et al., 2016). Here, we describe in detail the method for analysis of the elongation and the TIR1/AFB-Aux/IAA-dependent auxin response in hypocotyl segments as well as the determination of relative values of the cell wall pH.
January 5, 2018: Bio-protocol
Teresa Rojo Romanos, Leelee Ng, Roger Pocock
Animals use behavioral strategies to seek optimal environments. Population behavioral assays provide a robust means to determine the effect of genetic perturbations on the ability of animals to sense and respond to changes in the environment. Here, we describe a C. elegans population behavioral assay used to measure locomotory responses to changes in environmental oxygen (O2) and carbon dioxide (CO2) concentrations. These behavioral assays are high-throughput and enable examination of genetic, neuronal and circuit function...
January 5, 2018: Bio-protocol
Katarina Trajkovic, Hyunkyung Jeong, Dimitri Krainc
Quantitative analysis of proteins secreted from the cells poses a challenge due to their low abundance and the interfering presence of a large amount of bovine serum albumin (BSA) in the cell culture media. We established assays for detection of mutant huntingtin (mHtt) secreted from Neuro2A cell line stably expressing mHtt and rat primary cortical neurons by Western blotting. Our protocol is based on reducing the amounts of BSA in the media while maintaining cell viability and secretory potential, and concentrating the media prior to analysis by means of ultrafiltration...
January 5, 2018: Bio-protocol
Hellyeh Hamidi, Johanna Lilja, Johanna Ivaska
Cell adhesion to neighbouring cells and to the underlying extracellular matrix (ECM) is a fundamental requirement for the existence of multicellular organisms. As such, the formation, stability and dissociation of cell adhesions are subject to tight control in space and time and perturbations within the sophisticated adhesion machinery are associated with a variety of human pathologies. Here, we outline a simple protocol to monitor alterations in cell adhesion to the ECM, for example, following genetic manipulations or overexpression of a protein of interest or in response to drug treatment, using the xCELLigence real-time cell analysis (RTCA) system...
December 20, 2017: Bio-protocol
Damayanti Chakraborty, Masanaga Muto, Michael J Soares
In this protocol report, we describe a lentiviral gene delivery technique for genetic modification of the rat trophoblast cell lineage. Lentiviral packaged gene constructs can be efficiently and specifically delivered to the trophoblast cell lineage of the blastocyst. The consequences of 'gain-of-function' and 'loss-of-function' blastocyst manipulations can be evaluated with in vitro outgrowth assays or following transfer to pseudopregnant rats.
December 20, 2017: Bio-protocol
Loreto Abusleme, Bo-Young Hong, Anilei Hoare, Joanne E Konkel, Patricia I Diaz, Niki M Moutsopoulos
The oral microbiome has been implicated as a trigger for immune responsiveness in the oral cavity, particularly in the setting of the inflammatory disease periodontitis. The protocol presented here is aimed at characterizing the oral microbiome in murine models at steady state and during perturbations of immunity or physiology. Herein, we describe murine oral microbiome sampling procedures, processing of low biomass samples and subsequent microbiome characterization based on 16S rRNA gene sequencing.
December 20, 2017: Bio-protocol
Rubul Mout, Vincent M Rotello
In this protocol, we describe a method for direct cytosolic protein delivery that avoids endosomal entrapment of the delivered proteins. We achieved this by tagging the desired protein with an oligo glutamic acid tag (E-tag), and subsequently using carrier gold nanoparticles to deliver these E-tagged proteins. When E-tagged proteins and nanoparticles were mixed, they formed nanoassemblies, which got fused to cell membrane upon incubation and directly released the E-tagged protein into cell cytosol. We used this method to deliver a wide variety of proteins with different sizes, charges, and functions in various cell lines (Mout et al...
December 20, 2017: Bio-protocol
Shweta Varshney, Pamela Stanley
Notch signaling is an evolutionary conserved signaling pathway that plays an indispensable role during development, and in the maintenance of homeostatic processes, in a wide variety of tissues (Kopan, 2012; Hori et al., 2013). The multifaceted roles of Notch signaling are stringently regulated at different levels. One of the most important aspects of regulation is the binding of different Notch ligands to each Notch receptor (NOTCH1-NOTCH4). Canonical ligands Delta or Serrate (in Drosophila), and Delta-like (DLL1 and DLL4) or Jagged (JAG1 and JAG2) (in mammals), are transmembrane glycoproteins...
December 5, 2017: Bio-protocol
Jamie Jiang, Jessica W Bertol, Walid D Fakhouri
A major issue in developmental biology is to determine how embryonic tissues respond to molecular signals in a timely manner and given the position-restricted instructions during morphogenesis, of which Meckel's cartilage is a classical example. The ex-vivo explant model is a practical and convenient system that allows investigation of bone and cartilage responses to specific stimuli under a controlled manner that closely mimics the in vivo conditions. In this protocol, the explant model was applied to test whether Meckel's cartilage and surrounding tissues are responsive to the Endothelin1 (Edn1) signaling molecule and whether it would rescue the phenotype of genetic mutations...
December 5, 2017: Bio-protocol
Kendi Okuda, Neal Silverman
This protocol describes how to generate and harvest antibody-free L. amazonensis amastigotes, and how to infect adult Drosophila melanogaster with these parasites. This model recapitulates key aspects of the interactions between Leishmania amastigotes and animal phagocytes.
December 5, 2017: Bio-protocol
A Marieke Oudelaar, Damien J Downes, James O J Davies, Jim R Hughes
Chromosome conformation capture (3C) techniques are crucial to understanding tissue-specific regulation of gene expression, but current methods generally require large numbers of cells. This protocol describes two new low-input Capture-C approaches that can generate high-quality 3C interaction profiles from 10,000-20,000 cells, depending on the resolution used for analysis.
December 5, 2017: Bio-protocol
Yu Shu Xiao, Frieder Schöck, Nicanor González-Morales
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 dissection of IFM and analysis of sarcomeres using fluorescently tagged proteins.
November 20, 2017: Bio-protocol
Kaleeckal G Harikumar, Yan Yan, Ting-Hai Xu, Karsten Melcher, H Eric Xu, Laurence J Miller
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 tagged with bioluminescent Renilla luciferase (Rlu) as donor and yellow fluorescent protein (YFP) as acceptor were co-expressed in cells. This pair of donor and acceptor have an approximate Förster distance of 4...
November 20, 2017: Bio-protocol
Ting-Hai Xu, Yan Yan, Kaleeckal G Harikumar, Laurence J Miller, Karsten Melcher, H Eric Xu
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 co-associated Flag-tagged protein using Streptavidin MagBeads.
November 20, 2017: Bio-protocol
Yan Yan, Ting-Hai Xu, Kaleeckal G Harikumar, Laurence J Miller, Karsten Melcher, H Eric Xu
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 efficient cleavage of the transcription factor, allowing it to translocate to the nucleus and activate a luciferase reporter gene as measurement of the interactions. In this modified assay, we fused one copy of the membrane-spanning amyloid precursor protein (APP) C99 region to TEV site-rTA (C99-TEV site-rTA) and a second copy to TEV protease (C99-TEV) to analyze intramembrane C99-C99 interaction in live cells...
November 20, 2017: Bio-protocol
Ting-Hai Xu, Yan Yan, Kaleeckal G Harikumar, Laurence J Miller, Karsten Melcher, H Eric Xu
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 operator element, in which C99 C terminally fused to a reversed tetracyclin-inducible activator (rTA) transcriptional activator is expressed. Endogenous or transfected γ-secretase cleaves a C terminally fused rTA transcriptional activator from C99, allowing rTA to move to the nucleus to activate a luciferase reporter gene as a measurement for γ-secretase cleavage activity...
November 20, 2017: Bio-protocol
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