Volume: 10 Issue: 13 —BIO-PROTOCOL

Biochemistry

Protocol for Peptide Synthesis on Spectrally Encoded Beads for MRBLE-pep Assays Authors: Jamin B. Hein, Huy Q. Nguyen, Martha Cyert and Polly M. Fordyce, date: 07/05/2020, view: 810, Q&A: 0

Every living cell relies on signal transduction pathways comprised of protein-protein interactions (PPIs). In many cases, these PPIs are between a folded protein domain and a short linear motif (SLiM) within an unstructured region of a protein. As a result of this small interaction interface (3-10 amino acids), the affinities of SLiM-mediated ...

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Gas Chromatography Detection Protocol of Short-chain Fatty Acids in Mice Feces Authors: Chaozheng Zhang, An Liu, Tianshuang Zhang, Yi Li and Hua Zhao, date: 07/05/2020, view: 660, Q&A: 0

Short-chain fatty acids (SCFAs), which are formed mainly by bacteria fermenting undigested carbohydrates in the colon, they are based on the number of carbon atoms in the carbon chain. Organic fatty acids with less than 6 carbon atoms are called short-chain fatty acids. SCFAs are closely related to various aspects of the human body, so more and ...

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Preparation of Drosophila Polytene Chromosomes, Followed by Immunofluorescence Analysis of Chromatin Structure by Multi-fluorescence Correlations Authors: Terra M. Kuhn, Shawn C. Little and Maya Capelson, date: 07/05/2020, view: 572, Q&A: 0

Drosophila larval salivary gland polytene chromosome squashes have been used for decades to analyze genome-wide protein-binding patterns, transcriptional activation processes, and changes in chromatin structure at specific genetic loci. There have been many evolutions of the squashing protocol over the years, with sub-optimal ...

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In vitro Assessment of Pathogen Effector Binding to Host Proteins by Surface Plasmon Resonance Authors: Marina Franceschetti, Mark J. Banfield, Clare E. M. Stevenson and Juan Carlos De la Concepcion, date: 07/05/2020, view: 911, Q&A: 0

The mechanisms of virulence and immunity are often governed by molecular interactions between pathogens and host proteins. The study of these interactions has major implications on understanding virulence activities, and how the host immune system recognizes the presence of pathogens to initiate an immune response. Frequently, the association ...

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Biophysics

NMR waterLOGSY as An Assay in Drug Development Programmes for Detecting Protein-Ligand Interactions–NMR waterLOGSY Authors: Carole J. R. Bataille, Terence H. Rabbitts and Timothy D. W. Claridge, date: 07/05/2020, view: 593, Q&A: 0

In drug development programmes, multiple assays are needed for the determination of protein-compound interactions and evaluation of potential use in assays with protein-protein interactions. In this protocol we describe the waterLOGSY NMR method for confirming protein-ligand binding events.

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Cancer Biology

Isolation of Lipid Rafts from Cultured Mammalian Cells and Their Lipidomics Analysis Authors: Nigora Mukhamedova, Kevin Huynh, Hann Low, Peter J. Meikle and Dmitri Sviridov, date: 07/05/2020, view: 645, Q&A: 0

Lipid rafts are distinct liquid-ordered domains of plasma membranes of most eukaryotic cells providing platform for signaling pathways. Lipid composition of rafts is critical for their structural integrity and for regulation of signaling pathways originating from rafts. Here we provide a protocol to isolate lipid rafts from cultured human and ...

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Tyramide Signal-Amplified Immunofluorescence of MYCN and MYC in Human Tissue Specimens and Cell Line Cultures Authors: Johanna M. Schafer and Jennifer A. Pietenpol, date: 07/05/2020, view: 550, Q&A: 0

MYC family members, MYC, MYCN, and MYCL, are oncogenic transcription factors that regulate the expression of genes involved in normal development, cell growth, proliferation, metabolism, and survival. While MYC is amplified and/or overexpressed across a variety of tissue types, MYCN is often overexpressed in tumors of the nervous system ...

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Generation and Testing of Fluorescent Adaptable Simple Theranostic (FAST) Proteins Authors: Andrew S. Flies, Jocelyn M. Darby, Peter R. Murphy, Terry L. Pinfold, Amanda L. Patchett and Patrick R. Lennard, date: 07/05/2020, view: 945, Q&A: 0

This protocol provides a step-by-step method to create recombinant fluorescent fusion proteins that can be secreted from mammalian cell lines. This builds on many other recombinant protein and fluorescent protein techniques, but is among the first to harness fluorescent fusion proteins secreted directly into cell culture supernatant. This opens ...

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Immunology

Co-immunostaining of ICAM-1, ICAM-2, and CD31 in Mouse Kidney Glomeruli Author: Sun-sang J. Sung, date: 07/05/2020, view: 508, Q&A: 0

Glomerulonephritis (GN) is a common pathological condition in chronic kidney diseases that often leads to end stage renal failure. Mac-1 (CD11b/CD18)-mediated neutrophil, macrophage, and dendritic cell glomerular infiltration leading to cellular dysfunction and destruction is an important disease mechanism. The cellular distribution and dynamics ...

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Generation of T cells from Human and Nonhuman Primate Pluripotent Stem Cells Authors: Akhilesh Kumar, Saritha S. D’Souza, Gene Uenishi, Mi Ae Park, Jeong Hee Lee and Igor I. Slukvin, date: 07/05/2020, view: 593, Q&A: 0

Pluripotent stem cells (PSCs) have the potential to provide homogeneous cell populations of T cells that can be grown at a clinical scale and genetically engineered to meet specific clinical needs. OP9-DLL4, a stromal line ectopically expressing the Notch ligand Delta-like 4 (DLL4) is used to support differentiation of PSCs to T-lymphocytes. This ...

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Microbiology

Microscopy-based Methods for Rosetting Assay in Malaria Research Authors: Wenn-Chyau Lee and Laurent Rénia, date: 07/05/2020, view: 666, Q&A: 0

In malaria, rosetting phenomenon is a condition where a Plasmodium-infected erythrocyte stably adheres to at least an uninfected erythrocyte. This phenomenon that occurs in all species of human malaria parasite is likely to be an immune escape mechanism for the parasite. However, it has been associated with malaria pathogenesis, possibly ...

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Observing Nutrient Gradients, Gene Expression and Growth Variation Using the "Yeast Machine" Microfluidic Device Authors: Zoran S. Marinkovic, Clément Vulin, Mislav Acman, Xiaohu Song, Jean Marc Di Meglio, Ariel B. Lindner and Pascal Hersen, date: 07/05/2020, view: 496, Q&A: 0

The natural environment of microbial cells like bacteria and yeast is often a complex community in which growth and internal organization reflect morphogenetic processes and interactions that are dependent on spatial position and time. While most of research is performed in simple homogeneous environments (e.g., bulk liquid cultures), ...

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Neuroscience

The Discrete Paired-trial Variable-delay T-maze Task to Assess Working Memory in Mice Authors: Gian Marco Leggio, Sebastiano Alfio Torrisi and Francesco Papaleo, date: 07/05/2020, view: 457, Q&A: 0

Working memory abnormalities involving the prefrontal cortex (PFC) dramatically contribute to poor functional outcomes in patients with schizophrenia and still represent an unmet therapeutic need. Studies in rodents might provide essential tools to understand the mechanisms underlying PFC-dependent working memory dysfunctions, as well as precious ...

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Organotypic Slice Culture of the Embryonic Mouse Brain Authors: James M. Clegg and Thomas Pratt, date: 07/05/2020, view: 440, Q&A: 0

Organotypic slice culture is a powerful technique for exploring the embryonic development of the mammalian brain. In this protocol we describe a basic slice culture technique we have used for two sets of experiments: axon guidance transplant assays and bead culture assays.

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Plant Science

Methylation-sensitive Amplified Polymorphism as a Tool to Analyze Wild Potato Hybrids Authors: Nicolás Cara, Carlos F. Marfil, María V. Bertoldi and Ricardo W. Masuelli, date: 07/05/2020, view: 473, Q&A: 0

Methylation-Sensitive Amplification Polymorphism (MSAP) is a versatile marker for analyzing DNA methylation patterns in non-model species. The implementation of this technique does not require a reference genome and makes it possible to determine the methylation status of hundreds of anonymous loci distributed throughout the genome. In addition, ...

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Stem Cell

In vivo Mouse Mammary Gland Formation Authors: Heng Sun, Xin Zhang, Un In Chan, Sek Man Su, Sen Guo, Xiaoling Xu and Chuxia Deng, date: 07/05/2020, view: 519, Q&A: 0

For years, the mammary gland serves as a perfect example to study the self-renew and differentiation of adult stem cells, and the regulatory mechanisms of these processes as well. To assess the function of given genes and/or other factors on stemness of mammary cells, several In vitro assays were developed, such as mammospheres formation ...

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