Volume: 8 Issue: 20 —BIO-PROTOCOL

Biochemistry

Nuclear/Cytoplasmic Fractionation of Proteins from Caenorhabditis elegans Authors: Alejandro Mata-Cabana, Olga Sin, Renée I. Seinstra and Ellen A. A. Nollen, date: 10/20/2018, view: 3593, Q&A: 0

C. elegans is widely used to investigate biological processes related to health and disease. To study protein localization, fluorescently-tagged proteins can be used in vivo or immunohistochemistry can be performed in whole worms. Here, we describe a technique to localize a protein of interest at a subcellular level in C. elegans ...

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Cell Biology

Vascular Permeability Assay in Human Coronary and Mouse Brachiocephalic Arteries Authors: Liang Guo, Raquel Fernandez, Atsushi Sakamoto, Anne Cornelissen, Ka Hyun Paek, Parker J. Lee, Leah M. Weinstein, Carlos J. Collado-Rivera, Emanuel Harari, Robert Kutys, Torie S. Samuda, Nicole A. Singer, Matthew D. Kutyna, Frank D. Kolodgie, Renu Virmani and Aloke V. Finn, date: 10/20/2018, view: 2357, Q&A: 0

Coronary artery disease remains an important cause of morbidity and mortality. Previous work, including ours, has focused on the role of intraplaque hemorrhage, particularly from immature microvessel angiogenesis, as an important contributor to plaque progression via increases in vascular permeability leading to further intraplaque hemorrhage, ...

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Transcytosis Assay for Transport of Glycosphingolipids across MDCK-II Cells Authors: Maria Daniela Garcia-Castillo, Wayne I. Lencer and Daniel J.-F. Chinnapen, date: 10/20/2018, view: 2404, Q&A: 0

Absorption and secretion of peptide and protein cargoes across single-cell thick mucosal and endothelial barriers occurs by active endocytic and vesicular trafficking that connects one side of the epithelial or endothelial cell (the lumen) with the other (the serosa or blood). Assays that assess this pathway must robustly control for non-specific ...

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Murine Pharmacokinetic Studies Authors: Alix F. Leblanc, Kevin M. Huang, Muhammad Erfan Uddin, Jason T. Anderson, Mingqing Chen and Shuiying Hu, date: 10/20/2018, view: 5021, Q&A: 0

Murine pharmacokinetics (PK) represents the absorption, distribution, metabolism, and elimination of drugs from the body, which helps to guide clinical studies, ultimately resulting in more effective drug treatment. The purpose of this protocol is to describe a serial bleeding protocol, obtaining blood samples at six time points from single mouse ...

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Immunology

A Highly Sensitive Anion Exchange Chromatography Method for Measuring cGAS Activity in vitro Authors: Andreas Holleufer and Rune Hartmann, date: 10/20/2018, view: 2119, Q&A: 0

Cyclic GMP-AMP synthase (cGAS) is a pattern recognition receptor (PRR) that senses double stranded DNA (dsDNA) in the cytosol and this leads to the activation of stimulator of interferon genes (STING) via the secondary messenger 2’3’-cyclic GMP-AMP (2’3’-cGAMP). STING then recruits TANK binding kinase 1 (TBK-1) and this complex can phosphorylate ...

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Microbiology

Preparation and Purification of Proteins Secreted from Phytophthora sojae Authors: Yeqiang Xia, Yan Wang and Yuanchao Wang, date: 10/20/2018, view: 2478, Q&A: 1

Phytophthora sojae, the causal agent of soybean root and stem rot, is responsible for enormous economic losses in soybean production. P. sojae secrets various effectors to reprogram host immunity. The plant apoplastic space is a major battleground in plant-pathogen interactions. Here we describe a protocol for purification and ...

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HIV_GKO: A Tool to Assess HIV-1 Latency Reversal Agents in Human Primary CD4⁺ T Cells Authors: Emilie Battivelli and Eric Verdin, date: 10/20/2018, view: 2704, Q&A: 0

While able to suppress HIV replication in HIV infected individuals, combination antiretroviral therapy (ART) fails to eliminate viral latent reservoir, which consists in integrated transcriptional silenced HIV provirus. So far, identification of latently-infected cells has relied on activating cells to induce expression of HIV proteins which can ...

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Detection and Differentiation of Multiple Viral RNAs Using Branched DNA FISH Coupled to Confocal Microscopy and Flow Cytometry Authors: Nicholas van Buuren and Karla Kirkegaard, date: 10/20/2018, view: 2206, Q&A: 0

Due to the exceptionally high mutation rates of RNA-dependent RNA polymerases, infectious RNA viruses generate extensive sequence diversity, leading to some of the lowest barriers to the development of antiviral drug resistance in the microbial world. We have previously discovered that higher barriers to the development of drug resistance can be ...

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Rice Ragged Stunt Virus Propagation and Infection on Rice Plants Authors: Chao Zhang, Chaonan Shi, Dong Chen and Jianguo Wu, date: 10/20/2018, view: 2041, Q&A: 0

Virus inoculation is a basic experimental procedure to evaluate the resistance of a rice variety or a transgenic material upon virus infection. We recently demonstrated that Rice Ragged Stunt Virus (RRSV), an oryzavirus that is transmitted by brown planthopper (BPH), can suppress jasmonic acid-mediated antiviral defense through ...

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H1N1 Virus Production and Infection Authors: Binbin Zhao, Jiaoyu Shan, Rui Xiong, Ke Xu and Bin Li, date: 10/20/2018, view: 2112, Q&A: 0

Influenza A virus is a member of orthomyxoviridae family causing wide-spread infections in human respiratory tract. Mouse infection model is widely used in antiviral research and pathogenesis study against influenza A virus. Here, we report a protocol in infected mice with different virus doses and strains to explore how an inhibitor of ...

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Assessing Membrane Fluidity and Visualizing Fluid Membrane Domains in Bacteria Using Fluorescent Membrane Dyes Authors: Michaela Wenzel, Norbert O. E. Vischer, Henrik Strahl and Leendert W. Hamoen, date: 10/20/2018, view: 3258, Q&A: 0

Membrane fluidity is a key parameter of bacterial membranes that undergoes quick adaptation in response to environmental challenges and has recently emerged as an important factor in the antibacterial mechanism of membrane-targeting antibiotics. The specific level of membrane fluidity is not uniform across the bacterial cell membrane. Rather, ...

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Molecular Biology

Microtitre Plate Based Cell-SELEX Method Authors: Munish Shorie and Harmanjit Kaur, date: 10/20/2018, view: 2552, Q&A: 0

Aptamers have emerged as a novel category in the field of bioreceptors due to their wide applications ranging from biosensing to therapeutics. Several variations of their screening process, called SELEX have been reported which can yield sequences with desired properties needed for their final use. We report a facile microtiter plate-based ...

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Generation of Gene Knockout and Gene Replacement with Complete Removal of Full-length Endogenous Transcript Using CRISPR-Trap Authors: Jonas Mechtersheimer, Stefan Reber and Marc-David Ruepp, date: 10/20/2018, view: 3053, Q&A: 0

This protocol describes the application of the CRISPR-Trap from designing of the gene targeting strategy to validation of successfully edited clones that was validated on various human cell lines, among them human induced pluripotent stem cells (hiPSCs). The advantage of CRISPR-Trap over conventional approaches is the complete removal of any ...

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Neuroscience

Optical Clearing and Index Matching of Tissue Samples for High-resolution Fluorescence Imaging Using SeeDB2 Authors: Meng-Tsen Ke and Takeshi Imai, date: 10/20/2018, view: 4619, Q&A: 1

Tissue clearing techniques are useful for large-scale three-dimensional fluorescence imaging of thick tissues. However, high-resolution imaging deep inside tissues has been challenging, as it is extremely sensitive to light scattering and spherical aberrations. Here, we present a water-based optical clearing and mounting media, SeeDB2, which is ...

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Testing for Assortative Mating by Diet in Drosophila melanogaster Authors: Philip T Leftwich and Tracey Chapman, date: 10/20/2018, view: 1840, Q&A: 0

Experimental studies of the evolution of reproductive isolation in real time are a powerful way to reveal the way that fundamental processes, such as mate choice, initiate divergence. Mate choice, while frequently described in females, can occur in either sex, and can be affected by the genetics or environment of an individual. Here we describe ...

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Behavioral Evaluation of Seeking and Preference of Alcohol in Mice Subjected to Stress Authors: Ana Canseco-Alba, Norman Schanz, Hiroki Ishiguro, Qing-Rong Liu and Emmanuel S. Onaivi, date: 10/20/2018, view: 2155, Q&A: 0

The alcohol preference model is one of the most widely used animal models relevant to alcoholism. Stressors increase alcohol consumption. Here we present a protocol for a rapid and useful tool to test alcohol preference and stress-induced alcohol consumption in mice. In this model, animals are given two bottles, one with a diluted solution of ...

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Plant Science

Examining Autophagy in Plant by Transmission Electron Microscopy (TEM) Authors: Xiyin Zheng, Chenguang Zhao and Yule Liu, date: 10/20/2018, view: 2391, Q&A: 0

In plants, macroautophagy, here referred as autophagy, is a degradation pathway during which the double-membrane structure named autophagosome engulfs the cargo and then fuses with vacuole for material recycling.

To investigate the process of autophagy, transmission electron microscopy (TEM) was used to monitor the ultrastructure of ...

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Pneumatic Method to Measure Plant Xylem Embolism Authors: Paulo R. L. Bittencourt, Luciano Pereira and Rafael S. Oliveira, date: 10/20/2018, view: 2463, Q&A: 0

Embolism, the formation of air bubbles in the plant water transport system, has a major impact on plant water relations. Embolism formation in the water transport system of plants disrupts plant water transport capacity, impairing plant functioning and triggering plant mortality. Measuring embolism with traditional hydraulic methods is both ...

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Past Issues in 2018

Volume: 8 Issue: 20

Biochemistry

Cell Biology

Immunology

Microbiology

Molecular Biology

Neuroscience

Plant Science