Volume: 8 Issue: 17 —BIO-PROTOCOL

Biochemistry

Structural Analysis of Target Protein by Substituted Cysteine Accessibility Method Authors: Tetsuo Cai and Taisuke Tomita, date: 09/05/2018, view: 2603, Q&A: 0

Substituted Cysteine Accessibility Method (SCAM) is a biochemical approach to investigate the water accessibility or the spatial distance of particular cysteine residues substituted in the target protein. Protein topology and structure can be annotated by labeling with methanethiosulfonate reagents that specifically react with the cysteine ...

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Detection of Internal Matrix Targeting Signal-like Sequences (iMTS-Ls) in Mitochondrial Precursor Proteins Using the TargetP Prediction Tool Authors: Felix Boos, Timo Mühlhaus and Johannes M. Herrmann, date: 09/05/2018, view: 2966, Q&A: 0

Mitochondria contain hundreds of proteins which are encoded by the nuclear genome and synthesized in the cytosol from where they are imported into the organelle. Sorting signals encoded in the primary and secondary sequence of these proteins mediate the recognition of newly synthesized precursor proteins and their subsequent translocation through ...

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Cancer Biology

Cell Synchronization by Double Thymidine Block Authors: Guo Chen and Xingming Deng, date: 09/05/2018, view: 17916, Q&A: 1

Cell synchronization is widely used in studying mechanisms involves in regulation of cell cycle progression. Through synchronization, cells at distinct cell cycle stage could be obtained. Thymidine is a DNA synthesis inhibitor that can arrest cell at G1/S boundary, prior to DNA replication. Here, we present the protocol to synchronize cells at ...

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Developmental Biology

Human Endothelial Cell Spheroid-based Sprouting Angiogenesis Assay in Collagen Authors: Fabian Tetzlaff and Andreas Fischer, date: 09/05/2018, view: 7243, Q&A: 0

Angiogenesis, the formation of new blood vessels from pre-existing ones plays an important role during organ development, regeneration and tumor progression. The spheroid-based sprouting assay is a well-established and robust method to study the influence of genetic alterations or pharmacological compounds on capillary-like tube formation of ...

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Immunology

Using Stable Isotopes in Bone Marrow Derived Macrophage to Analyze Metabolism Authors: Chih-Wei Ko, Daniel Counihan, David DeSantis, Zach Sedor-Schiffhauer, Michelle Puchowicz and Colleen M Croniger, date: 09/05/2018, view: 2690, Q&A: 0

Using gas chromatography mass spectrometry (GC-MS) to analyze the citric acid cycle (CAC) and related intermediates (such as glutamate, glutamine, GABA, and aspartate) is an analytical approach to identify unexpected correlations between apparently related and unrelated pathways of energy metabolism. Intermediates can be as expressed as their ...

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Microbiology

Artificial Inoculation of Epichloë festucae into Lolium perenne, and Visualisation of Endophytic and Epiphyllous Fungal Growth Authors: Yvonne Becker, Kimberly A. Green, Barry Scott and Matthias Becker, date: 09/05/2018, view: 3452, Q&A: 0

Natural hosts for the fungal endophyte Epichloë festucae include Festuca rubra (fine fescue) and Festuca trachyphylla (hard fescue). Some strains also form stable associations with Lolium perenne (perennial ryegrass). L. perenne is a suitable host to study fungal endophyte–grass interactions, such as endophytic ...

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Analysis of the Effect of Sphingomyelinase on Rubella Virus Infectivity in Two Cell Lines Authors: Noriyuki Otsuki, Masafumi Sakata, Yoshio Mori, Kiyoko Okamoto and Makoto Takeda, date: 09/05/2018, view: 2429, Q&A: 0

Rubella is a mildly contagious disease characterized by low-grade fever and a morbilliform rash caused by the rubella virus (RuV). Viruses often use cellular phospholipids for infection. We studied the roles of cellular sphingomyelin in RuV infection. Treatment of cells with sphingomyelinase (SMase) inhibited RuV infection in rabbit kidney-derived ...

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Soluble and Solid Iron Reduction Assays with Desulfitobacterium hafniense Authors: Lucrezia Comensoli, Julien Maillard, Wafa M. Kooli, Pilar Junier and Edith Joseph, date: 09/05/2018, view: 2329, Q&A: 0

There is a pressing need to develop sustainable and efficient methods to protect and stabilize iron objects. To develop a conservation-restoration method for corroded iron objects, this bio-protocol presents the steps to investigate reductive dissolution of ferric iron and biogenic production of stabilizing ferrous iron minerals in the strict ...

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Selective Isolation of Retroviruses from Extracellular Vesicles by Intact Virion Immunoprecipitation Authors: Tyler Milston Renner, Kasandra Bélanger and Marc-André Langlois, date: 09/05/2018, view: 2808, Q&A: 0

There exists a wide variety of techniques to isolate and purify viral particles from cell culture supernatants. However, these techniques vary greatly in ease of use, purity, yield and impact on viral structural integrity. Most importantly, it is becoming evident that secreted extracellular vesicles (EVs) co-purify with retroviruses using nearly ...

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Molecular Biology

A Quantitative Heterokaryon Assay to Measure the Nucleocytoplasmic Shuttling of Proteins Authors: François McNicoll and Michaela Müller-McNicoll, date: 09/05/2018, view: 3856, Q&A: 0

Many proteins appear exclusively nuclear at steady-state but in fact shuttle continuously back and forth between the nucleus and the cytoplasm. For example, nuclear RNA-binding proteins (RBPs) often accompany mRNAs to the cytoplasm, where they can regulate subcellular localization, translation and/or decay of their cargos before shuttling back to ...

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Enzymatic Synthesis and Fractionation of Fluorescent PolyU RNAs Authors: Christian Beren, Katherine N. Liu, Lisa L. Dreesens, Charles M. Knobler and William M. Gelbart, date: 09/05/2018, view: 2006, Q&A: 0

The physical properties of viral-length polyuridine (PolyU) RNAs, which cannot base-pair and form secondary structures, are compared with those of normal-composition RNAs, composed of comparable numbers of each of A, U, G and C nucleobases. In this protocol, we describe how to synthesize fluorescent polyU RNAs using the enzyme polynucleotide ...

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Dual Fluorescence Reporter Based Analytical Flow Cytometry for miRNA Induced Regulation in Mammalian Cells Authors: Nicolas Lemus-Diaz, Liezel Tamon and Jens Gruber, date: 09/05/2018, view: 5535, Q&A: 0

MicroRNA-induced gene regulation is a growing field in basic and translational research. Examining this regulation directly in cells is necessary to validate high-throughput data originated from RNA sequencing technologies. For this several studies employ luciferase-based reporters that usually measure the whole cell population, which comes with ...

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Neuroscience

Phagocytosis Assay for α-Synuclein Fibril Uptake by Mouse Primary Microglia Authors: Cuilian Du, Feifan Zhang and Claire Xi Zhang, date: 09/05/2018, view: 2589, Q&A: 0

Microglia are professional phagocytes in the brain and deficiency in their phagocytic activity plays an important role in Parkinson’s disease. This protocol mainly describes the phagocytosis assay for uptake of α-synuclein preformed fibrils, a pathologic form of α-synuclein, by primary microglia.

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Pentylenetetrazole (PTZ)-induced Convulsion Assay to Determine GABAergic Defects in Caenorhabditis elegans Authors: Shruti Thapliyal and Kavita Babu, date: 09/05/2018, view: 2545, Q&A: 0

Pentylenetetrazole (PTZ) is a GABA_A receptor antagonist and is used to monitor presynaptic defects in the release of the inhibitory neurotransmitter GABA. PTZ is a competitive inhibitor of GABA, and prevents binding of GABA on the GABA_A receptors present on the surface of muscle. In the absence of GABA binding, the excitatory ...

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Shock-probe Defensive Burying Test to Measure Active versus Passive Coping Style in Response to an Aversive Stimulus in Rats Authors: Elizabeth A. Fucich and David A. Morilak, date: 09/05/2018, view: 2411, Q&A: 0

Maladaptive avoidance behaviors are seen in many stress-related psychiatric illnesses. Patients with these illnesses favor passive, avoidant coping strategies rather than adaptive, active coping strategies. Preclinically, coping strategy can be measured in rats using the shock-probe defensive burying test, wherein rats receive a shock from an ...

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Studying the Mechanisms of Developmental Vocal Learning and Adult Vocal Performance in Zebra Finches through Lentiviral Injection Authors: Zhimin Shi, Ofer Tchernichovski and XiaoChing Li, date: 09/05/2018, view: 2720, Q&A: 0

Here we provide a detailed step-by-step protocol for using lentivirus to manipulate miRNA expression in Area X of juvenile zebra finches and for analyzing the consequences on song learning and song performance. This protocol has four parts: 1) making the lentiviral construct to overexpress miRNA miR-9; 2) packaging the lentiviral vector; 3) ...

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Plant Science

In planta Transcriptome Analysis of Pseudomonas syringae Authors: Tatsuya Nobori and Kenichi Tsuda, date: 09/05/2018, view: 4315, Q&A: 0

Profiling bacterial transcriptome in planta is challenging due to the low abundance of bacterial RNA in infected plant tissues. Here, we describe a protocol to profile transcriptome of a foliar bacterial pathogen, Pseudomonas syringae pv. tomato DC3000, in the leaves of Arabidopsis thaliana at an early stage of ...

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Enzymatic Assays and Enzyme Histochemistry of Tuta absoluta Feeding on Tomato Leaves Authors: Rim Hamza, José P. Beltrán and Luis A. Cañas, date: 09/05/2018, view: 2995, Q&A: 0

Enzymes play a key role in insect-plant relationships. For a better understanding of these interactions, we analyzed Tuta absoluta digestive enzymes. Here, we describe a detailed protocol for the detection of trypsin and papain-like enzymes in Tuta absoluta larvae by enzyme histochemistry. This assay uses frozen and unfixed ...

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Stem Cell

Investigating Neural Stem Cell and Glioma Stem Cell Self-renewal Potential Using Extreme Limiting Dilution Analysis (ELDA) Authors: Hong PT Nguyen, Paul M Daniel, Gulay Filiz and Theo Mantamadiotis, date: 09/05/2018, view: 3622, Q&A: 0

Glioma stem cells (GSC) grown as neurospheres exhibit similar characteristics to neural stem cells (NSC) grown as neurospheres, including the ability to self-renew and differentiate. GSCs are thought to play a role in cancer initiation and progression. Self-renewal potential of GSCs is thought to reflect many characteristics associated with ...

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Microarray, IPA and GSEA Analysis in Mice Models Authors: Stephanie N. Oprescu, Katharine A. Horzmann, Feng Yue, Jennifer L. Freeman and Shihuan Kuang, date: 09/05/2018, view: 4316, Q&A: 0

This protocol details a method to analyze two tissue samples at the transcriptomic level using microarray analysis, ingenuity pathway analysis (IPA) and gene set enrichment analysis (GSEA). Methods such as these provide insight into the mechanisms underlying biological differences across two samples and thus can be applied to interrogate a variety ...

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Past Issues in 2018

Volume: 8 Issue: 17

Biochemistry

Cancer Biology

Developmental Biology

Immunology

Microbiology

Molecular Biology

Neuroscience

Plant Science

Stem Cell