Volume: 10 Issue: 2 —BIO-PROTOCOL

Biochemistry

Ribonucleoprotein Immunoprecipitation (RIP) Analysis Authors: Jennifer L. Martindale, Myriam Gorospe and Maria L. Idda, date: 01/20/2020, view: 1217, Q&A: 0

RNAs and RNA-binding proteins (RBPs) can interact dynamically in ribonucleoprotein (RNP) complexes that play important roles in controlling gene expression programs. One of the powerful ways to investigate changes in the association of RNAs with an RBP of interest is by immunoprecipitation (IP) analysis of native RNPs. RIP (RNP ...

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Protocols for Processing and Interpreting cryoEM Data Using Bsoft: A Case Study of the Retinal Adhesion Protein, Retinoschisin Author: J. Bernard Heymann, date: 01/20/2020, view: 813, Q&A: 0

The goal of cryoEM is to determine the structures of biomolecules from electron micrographs. In many cases the processing is straightforward and can be handled with routine protocols. In other cases, the properties and behavior of the specimen require adaptions to properly interpret the data. Here I describe the protocols for examining the higher ...

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Biophysics

Studying Protein Aggregation in the Context of Liquid-liquid Phase Separation Using Fluorescence and Atomic Force Microscopy, Fluorescence and Turbidity Assays, and FRAP Authors: W. Michael Babinchak and Witold K. Surewicz, date: 01/20/2020, view: 1262, Q&A: 0

Liquid-liquid phase separation (LLPS) underlies the physiological assembly of many membrane-less organelles throughout the cell. However, dysregulation of LLPS may mediate the formation of pathological aggregates associated with neurodegenerative diseases. Here, we present complementary experimental approaches to study protein aggregation within ...

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Characterizing the Two-photon Absorption Properties of Fluorescent Molecules in the 680-1300 nm Spectral Range Authors: Mikhail Drobizhev, Rosana S Molina and Thomas E Hughes, date: 01/20/2020, view: 763, Q&A: 0

Two-photon laser scanning microscopy (2PLSM) is a state-of-the-art technique used for non-invasive imaging deep inside the tissue, with high 3D resolution, minimal out-of-focus photodamage, and minimal autofluorescence background. For optimal application of fluorescent probes in 2PLSM, their two-photon absorption (2PA) spectra, expressed in ...

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Cancer Biology

Isolation of Stem Cells, Endothelial Cells and Pericytes from Human Infantile Hemangioma Authors: Lan Huang and Joyce Bischoff, date: 01/20/2020, view: 799, Q&A: 0

Infantile hemangioma (IH) is a vascular tumor noted for its excessive blood vessel formation during infancy, glucose-transporter-1 (GLUT1)-positive staining of the blood vessels, and its slow spontaneous involution over several years in early childhood. For most children, IH poses no serious threat because it will eventually involute, but a subset ...

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Bone-in-culture Array to Model Bone Metastasis in ex vivo Condition Authors: Hai Wang and Xiang H.-F. Zhang, date: 01/20/2020, view: 887, Q&A: 0

Bone is the most frequently affected organ by metastases of breast cancer and prostate cancer. Our knowledge on bone metastasis is extremely limited due to the lack of potent and efficient experimental models. We developed the “Bone-In-Culture Array (BICA)” platform to model the bone colonization of cancer cells in ex vivo cultures. The ...

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Immunology

Quantifying HIV-1-Mediated Gut CD4⁺ T Cell Death in the Lamina Propria Aggregate Culture (LPAC) Model Authors: Stephanie M. Dillon, Kejun Guo, Moriah J. Castleman, Mario L. Santiago and Cara C. Wilson, date: 01/20/2020, view: 849, Q&A: 0

Gut CD4 T cells are major targets of HIV-1 and are massively depleted early during infection. To better understand the mechanisms governing HIV-1-mediated CD4 T cell death, we developed the physiologically-relevant Lamina Propria Aggregate Culture (LPAC) model. The LPAC model is ideal for studying CD4 T cell death induced by clinically-relevant ...

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Automated Analysis of Cell Surface Ruffling: Ruffle Quantification Macro Authors: Nicholas D. Condon, Jennifer L. Stow and Adam A. Wall, date: 01/20/2020, view: 844, Q&A: 1

Cell surface protrusions include F-actin rich, wave-like ruffles that are erected transiently in response to stimuli and during cell migration. Macrophages are innate immune cells that ruffle constitutively and more dramatically in cells activated by pathogens. Dorsal ruffles and their resulting macropinosomes are key sites for environmental ...

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Microbiology

Sulfatase Assay to Determine Influence of Plants on Microbial Activity in Soil Authors: Anna Koprivova, Achim Schmalenberger and Stanislav Kopriva, date: 01/20/2020, view: 1139, Q&A: 0

Sulfatase activity is often used as a measure of the activity of soil microorganisms. It is thus a suitable tool to investigate the response of microbes to plants. Here we present a method to determine the influence of various Arabidopsis genotypes on the function of soil microbiota using the sulfatase as a quantitative measure. We grew ...

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A Radioactive-free Kinase Inhibitor Discovery Assay Against the Trypanosoma brucei Glycogen Synthase Kinase-3 short (TbGSK-3s) Authors: Antonia Efstathiou and Despina Smirlis, date: 01/20/2020, view: 625, Q&A: 0

The identification of small molecules possessing inhibitory activity in vitro, against a given target kinase, is the first step in the drug discovery process. Herein, we describe a non radioactive protocol using luciferase-based ATP assay for the identification of inhibitors for the short isoform of the Trypanosoma brucei’s ...

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Measurement of the Promoter Activity in Escherichia coli by Using a Luciferase Reporter Authors: Yuki Yamanaka, Hiroki Watanabe, Erika Yamauchi, Yukari Miyake and Kaneyoshi Yamamoto, date: 01/20/2020, view: 812, Q&A: 0

The reporter system is widely used technique for measuring promoter activity in bacterial cells. Until now, a number of reporter system have been developed, but the bioluminescent reporter constructed from the bacterial luciferase genes is one of the useful systems for measuring in vivo dynamics of gene expression. The introduced ...

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Study of Microbial Extracellular Vesicles: Separation by Density Gradients, Protection Assays and Labelling for Live Tracking Authors: Carolina Coelho, Raghav Vij, Daniel Q. Smith, Nathan R. Brady, Anne Hamacher-Brady and Arturo Casadevall, date: 01/20/2020, view: 912, Q&A: 0

Extracellular vesicles (EVs) are produced by all domains of life including Bacteria, Archaea and Eukarya. EVs are critical for cellular physiology and contain varied cargo: virulence factors, cell wall remodeling enzymes, extracellular matrix components and even nucleic acids and metabolites. While various protocols for isolating EVs have been ...

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Neuroscience

Protocol for Measuring Free (Low-stress) Exploration in Rats Authors: Wojciech Pisula and Klaudia Modlinska, date: 01/20/2020, view: 1036, Q&A: 0

Research on exploratory behavior plays a key part in behavioral science. Studying exploratory behavior of laboratory rodents may provide important data about many developmental and neurobiological processes occurring in animal ontogenesis. The proposed protocol for measuring the free (low-stress) exploration behavior in rats is straightforward, ...

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A Single Test to Study Social Behavior and Repetitive Self-grooming in Mice Authors: Niraj V. Lawande, Ammar L. Ujjainwala and Catherine A. Christian, date: 01/20/2020, view: 893, Q&A: 0

The ability to recognize and interact with members of the same species is essential for social communication. Investigating the neural substrates of social interest and recognition may offer insights into the behavioral differences present in disorders affecting social behavior. Assays used to study social interest in rodents include the 3-chamber ...

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A Simple and Efficient Method for Concomitant Isolation and Culture of Enriched Astroglial and Microglial Cells from the Rat Spinal Cord Authors: Pooja Shree Mishra and Trichur R Raju, date: 01/20/2020, view: 1150, Q&A: 0

Investigations into glial biology have contributed substantially in understanding the physiology and pathology of the nervous system. However, intricacies of the neuron-glial and glial-glial interactions in vivo present significant challenges while delineating the individual cell-type contributions, thus making the in vitro ...

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Past Issues in 2020

Volume: 10 Issue: 2

Biochemistry

Biophysics

Cancer Biology

Immunology

Microbiology

Neuroscience