Volume: 10 Issue: 1 —BIO-PROTOCOL

Biophysics

Whole-cell and Perforated Patch-clamp Recordings from Acutely-isolated Murine Sino-atrial Node Cells Authors: Qadeer Aziz, Muriel Nobles and Andrew Tinker, date: 01/05/2020, view: 3237, Q&A: 0

Cardiac pacemaker cells of the sino-atrial node are responsible for the initiation of the heart beat and express an array of ion channels. The patch-clamp technique is the gold standard method for investigating the function of ion channels expressed in electrically active cells. Conventional whole-cell and perforated patch-clamp techniques can be ...

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Cancer Biology

Analysis of Random Migration of Cancer Cells in 3D Authors: Sai P. Visweshwaran and Alexis Gautreau, date: 01/05/2020, view: 3034, Q&A: 0

The ability of cancer cells to migrate through a complex three-dimensional (3D) environment is a hallmark event of cancer metastasis. Therefore, an in vitro migration assay to evaluate cancer cell migration in a 3D setting is valuable to examine cancer progression. Here, we describe such a simple migration assay in a 3D ...

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Cell Biology

Acute Isolation of Cells from Murine Sino-atrial Node Authors: Qadeer Aziz, Muriel Nobles and Andrew Tinker, date: 01/05/2020, view: 2634, Q&A: 0

The cardiac conduction system allows the synchronized propagation of electrical activity through heart muscle. This is initiated by the spontaneous activity of the specialized pacemaker cells of the sino-atrial node (SAN). The SAN region underlies automaticity in mammals and therefore has a crucial role in the pathogenesis of cardiac disorders ...

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Steady-state and Flux-based Trehalose Estimation as an Indicator of Carbon Flow from Gluconeogenesis or Glycolysis Authors: Ritu Gupta and Sunil Laxman, date: 01/05/2020, view: 2594, Q&A: 0

Trehalose (and glycogen) is a major storage carbohydrate in many cells, including S. cerevisiae. Typically, trehalose (a disaccharide of glucose) is synthesized and stored through gluconeogenesis. However, trehalose can also be made directly from glucose, if glucose-6-phosphate is channeled away from glycolysis or pentose phosphate ...

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Developmental Biology

RNA Interactome Identification via RNA-BioID in Mouse Embryonic Fibroblasts Authors: Joyita Mukherjee, Mirita Franz-Wachtel, Boris Maček and Ralf-Peter Jansen, date: 01/05/2020, view: 2884, Q&A: 0

Cytoplasmic localization of mRNAs is common to all organisms and serves the spatial expression of genes. Cis-acting RNA signals (mostly found in the mRNA’s 3'-UTR), called zipcodes recruit trans-acting RNA-binding proteins that facilitate the localization of the mRNA. UV-cross-linking or affinity purification has been applied to identify such ...

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Microbiology

A Microbial Bioassay for Direct Contact Assessment of Soil Toxicity Based on Oxygen Consumption of Sulfur Oxidizing Bacteria Authors: Ebenezer Ashun, Umair Ali Toor, Heonseop Eom and Sang-Eun Oh, date: 01/05/2020, view: 2165, Q&A: 0

A new direct contact assessment of soil toxicity using sulfur oxidizing bacteria (SOB) is proposed for analyzing the toxicity of soils. The proposed method is based on the ability of SOB to oxidize elemental sulfur to sulfuric acid in the presence of oxygen. Since sulfate ions are produced from sulfur by SOB oxidation activity, changes in ...

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A Yeast Chromatin-enriched Fractions Purification Approach, yChEFs, from Saccharomyces cerevisiae Authors: Abel Cuevas-Bermúdez, Ana I. Garrido-Godino, Francisco Gutiérrez-Santiago and Francisco Navarro, date: 01/05/2020, view: 2107, Q&A: 0

We have adapted a previous procedure and improved an approach that we named yChEFs (yeast Chromatin Enriched Fractions) for purifying chromatin fractions. This methodology allows the easy, reproducible and scalable recovery of proteins associated with chromatin. By using yChEFs, we bypass subcellular fractionation requirements involved when using ...

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Unbiased and Tailored CRISPR/Cas gRNA Libraries by Synthesizing Covalently-closed-circular (3Cs) DNA Authors: Martin Wegner, Koraljka Husnjak and Manuel Kaulich, date: 01/05/2020, view: 4673, Q&A: 0

Simplicity, efficiency and versatility of the CRISPR/Cas system greatly contributed to its rapid use in a broad range of fields. Applications of unbiased CRISPR/Cas screenings are increasing and thus there is a growing need for unbiased and tailored CRISPR/Cas gRNA libraries. Conventional methods for gRNA library generation apply PCR and cloning ...

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Isolation and Characterization of Live Yeast Cells from Ancient Clay Vessels Authors: Tzemach Aouizerat, Aren M. Maeir, Yitzhak Paz, Yuval Gadot, Amir Szitenberg, Sivan Alkalay-Oren, Shunit Coppenhagen-Glazer, Michael Klutstein and Ronen Hazan, date: 01/05/2020, view: 3273, Q&A: 0

Ancient fermented food has been studied mainly based on residue analysis and recipes and reconstruction attempts were performed using modern domesticated yeast. Furthermore, microorganisms which participated in fermentation were studied using ancient-DNA techniques. In a recent paper, we presented a novel approach based on the hypothesis that ...

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Molecular Biology

High-throughput Site-directed Scanning Mutagenesis Using a Two-fragment PCR Approach Authors: Franziska M. Heydenreich, Tamara Miljuš, Dalibor Milić and Dmitry B. Veprintsev, date: 01/05/2020, view: 3091, Q&A: 0

Site-directed scanning mutagenesis is a useful tool applied in studying protein function and designing proteins with new properties, such as increased stability or enzymatic activity. Creating a systematic library of hundreds of site-directed mutants is still a demanding and expensive task. The established protocols for making such libraries ...

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Neuroscience

Laser Capture Micro-dissection (LCM) of Neonatal Mouse Forebrain for RNA Isolation Authors: Achira Roy, Mei Deng, Kimberly A. Aldinger, Ian A. GLass and Kathleen J. Millen, date: 01/05/2020, view: 3717, Q&A: 0

Precise and reproducible isolation of desired cell types or layers from heterogeneous tissues is crucial to analyze specific gene profiles and molecular interactions in vivo. Forebrain is the core site of higher functions, like cognition and memory consolidation. It is composed of heterogeneous and distinct cell types, interconnected to ...

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The Mouse Gambling Task: Assessing Individual Decision-making Strategies in Mice Authors: Elsa Pittaras, Arnaud Rabat and Sylvie Granon, date: 01/05/2020, view: 2407, Q&A: 0

Decision-making is a complex cognitive process which consists of choosing one option among several alternatives. In humans, this process is featured in the Iowa gambling task (IGT), a decision-making task that mimics real life situations by reproducing uncertain conditions based on probabilistic rewards or penalties (see Background). Several ...

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Assessing Rough-and-tumble Play Behavior in Juvenile Rats Authors: Jonathan W. VanRyzin, Ashley E. Marquardt and Margaret M. McCarthy, date: 01/05/2020, view: 2578, Q&A: 0

Play is a complex social behavior that is highly conserved across mammals. In most species, males engage in more frequent and vigorous play as juveniles than females, which reflects subtle yet impactful sex differences in brain circuitry and development. In this protocol, we describe a behavioral testing paradigm to assess social play in male and ...

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Plant Science

Measurement of Chloroplastic NAD Kinase Activity and Whole Tissue NAD Kinase Assay Authors: Yuuma Ishikawa, Maki Kawai-Yamada and Shin-nosuke Hashida, date: 01/05/2020, view: 2552, Q&A: 0

Nicotinamide adenine dinucleotide phosphate (NADP) synthesis requires nicotinamide adenine dinucleotide (NAD) kinase activity, substrate NAD and ATP. The NAD kinase responds to various environmental stimuli and its activity is regulated via various regulatory pathways, such as Ca²⁺-dependent and redox-dependent signals. Conventional in ...

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Visualization of Actin Organization and Quantification in Fixed Arabidopsis Pollen Grains and Tubes Authors: Xiaolu Qu, Qiannan Wang, Haiyan Wang and Shanjin Huang, date: 01/05/2020, view: 2460, Q&A: 0

Although it is widely accepted that actin plays an important role in regulating pollen germination and pollen tube growth, how actin exactly performs functions remains incompletely understood. As the function of actin is dictated by its spatial organization, it is the key to reveal how exactly actin distributes in space in pollen cells. Here we ...

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Past Issues in 2020

Volume: 10 Issue: 1

Biophysics

Cancer Biology

Cell Biology

Developmental Biology

Microbiology

Molecular Biology

Neuroscience

Plant Science