Volume: 10 Issue: 7 —BIO-PROTOCOL

Biochemistry

Preparation, FPLC Purification and LC-FT-ICR-MS of Proteins Authors: Tyler B. Johnson, Jiri Adamec and Paul Blum, date: 04/05/2020, view: 2047, Q&A: 0

High magnetic field Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometers provide extremely high mass resolution (resolving power of ~200,000 at 400 m/z) protein detection across a broad mass range, enabling analysis of fine structure of isotopic peak clusters that is missed in other types of mass spectrometers. The protocol ...

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Determination of Flavin Potential in Proteins by Xanthine/Xanthine Oxidase Method Authors: Elena Maklashina and Gary Cecchini, date: 04/05/2020, view: 1899, Q&A: 0

This protocol describes a simple xanthine/xanthine oxidase enzymatic equilibration method for determination of the redox potential of a flavin. As an example of the use of this method, we determine the reduction potential of the covalently bound FAD cofactor (E_m = -55 mV) in the SdhA flavoprotein subunit of succinate ...

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Measurement of Protein-Protein Interactions through Microscale Thermophoresis (MST) Authors: Magnez Romain, Bryan Thiroux, Morgane Tardy, Bruno Quesnel and Xavier Thuru, date: 04/05/2020, view: 2580, Q&A: 0

The binding interactions of PD-1 and PD-L1 have been studied by surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC) over the past few years, but these investigations resulted in controversy regarding the values of the dissociation constant (K_d) (Freeman et al., 2000). MST is a powerful new method for the ...

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Spectrophotometric Assessment of Heme Oxygenase-1 Activity in Leishmania-infected Macrophages Authors: Moumita Basu, Shriya Saha and Anindita Ukil, date: 04/05/2020, view: 2074, Q&A: 0

Heme oxygenase-1 (HO-1) is a stress responsive enzyme that metabolizes heme and releases free iron, carbon monoxide (CO), and biliverdin (BV), which rapidly undergoes conversion to bilirubin (BL). Estimation of bilirubin is the basis of HO-1 assay. HO-1 activity is widely employed to determine antioxidant response of cells under different ...

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Quantitative Determination of Ca²⁺-binding to Ca²⁺-sensor Proteins by Isothermal Titration Calorimetry Authors: Seher Abbas and Karl-Wilhelm Koch, date: 04/05/2020, view: 2272, Q&A: 0

Diverse and complex molecular recognitions are central elements of signal transduction cascades. The strength and nature of these interaction modes can be determined by different experimental approaches. Among those, Isothermal titration calorimetry (ITC) offers certain advantages by providing binding constants and thermodynamic parameters from ...

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Cancer Biology

In vivo Tumor Growth and Spontaneous Metastasis Assays Using A549 Lung Cancer Cells Authors: Lei Qi, Teresa Knifley, Dava W. Piecoro, Piotr Rychahou, Jianrong Wu, Kathleen L. O’Connor and Min Chen, date: 04/05/2020, view: 2886, Q&A: 0

Metastasis accounts for the majority of cancer related deaths. The genetically engineered mouse (GEM) models and cell line-based subcutaneous and orthotopic mouse xenografts have been developed to study the metastatic process. By using lung cancer cell line A549 as an example, we present a modified protocol to establish the cell line-based ...

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Cell Biology

Flow Cytometry Measurement of Glucocerebrosidase Activity in Human Monocytes Authors: Laura P. Hughes, Glenda M. Halliday and Nicolas Dzamko, date: 04/05/2020, view: 2542, Q&A: 0

Glucocerebrosidase (GCase) is an important enzyme for the metabolism of glycolipids. GCase enzyme deficiency is implicated in human disease and the efficient measurement of GCase activity is important for evaluating the efficacy of therapeutics targeting this enzyme. Existing approaches to measure GCase activity include whole blood mass ...

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Isolation, Purification and Characterization of Exosomes from Fibroblast Cultures of Skeletal Muscle Authors: Diantha van de Vlekkert, Xiaohui Qiu, Ida Annunziata and Alessandra d'Azzo, date: 04/05/2020, view: 2559, Q&A: 0

Exosomes are dynamic nanovesicles secreted by virtually all cells and are present in all biological fluids. Given their highly heterogeneous content exosomes have been implicated in many physiological and pathological processes that they exert by influencing cell-cell and cell-ECM communication. In recent years an increasing number of methods have ...

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Fractionation of Ovarian Follicles and in vitro Oocyte Maturation and Ovulation Assay of Ciona intestinalis Type A Authors: Shin Matsubara, Akira Shiraishi, Tomohiro Osugi, Tsuyoshi Kawada and Honoo Satake, date: 04/05/2020, view: 1957, Q&A: 0

Ascidians are the closest living relatives of vertebrates (Delsuc et al., 2006; Satoh et al., 2014) and are important for the evolutionary study of the ovarian follicle development including oocyte maturation and ovulation. However, neither the endogenous factors nor the molecular mechanisms underlying the oocyte maturation and ...

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Developmental Biology

Bimolecular Fluorescence Complementation (BiFC) for Studying Sarcomeric Protein Interactions in Drosophila Authors: Océane Marescal, Frieder Schöck and Nicanor González-Morales, date: 04/05/2020, view: 2472, Q&A: 0

Protein-protein interactions in Drosophila myofibrils are essential for their function and formation. Bimolecular Fluorescence Complementation (BiFC) is an effective method for studying protein interactions and localization. BiFC relies on the reconstitution of a monomeric fluorescent protein from two half-fragments when in proximity. Two ...

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Microbiology

Conjugation Protocol Optimised for Roseburia inulinivorans and Eubacterium rectale Authors: Paul O. Sheridan, Jennifer C. Martin and Karen P. Scott, date: 04/05/2020, view: 1963, Q&A: 0

Roseburia and Eubacterium species of the human gut microbiota play an important role in the maintaince of human health, partly by producing butyrate, the main energy source of our colonic epithelial cells. However, our knowledge of the biochemistry and physiology of these bacteria has been limited by a lack of genetic ...

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Molecular Biology

Preparation of a Bacteriophage T4-based Prokaryotic-eukaryotic Hybrid Viral Vector for Delivery of Large Cargos of Genes and Proteins into Human Cells Authors: Jingen Zhu, Pan Tao, Marthandan Mahalingam and Venigalla B. Rao, date: 04/05/2020, view: 1694, Q&A: 0

A viral vector that can safely and efficiently deliver large and diverse molecular cargos into cells is the holy grail of curing many human diseases. Adeno-associated virus (AAV) has been extensively used but has a very small capacity. The prokaryotic virus T4 has a large capacity but lacks natural mechanisms to enter mammalian cells. Here, we ...

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Measuring cAMP Specific Phosphodiesterase Activity: A Two-step Radioassay Authors: Connor M. Blair, Jiayue Ling and George S. Baillie, date: 04/05/2020, view: 1288, Q&A: 0

Cyclic nucleotide degrading phosphodiesterase (PDE) enzymes are crucial to the fine tuning of cAMP signaling responses, playing a pivotal role in regulating the temporal and spatial characteristics of discrete cAMP nanodomains and hence the activity of cAMP-effector proteins. As a consequence of orchestrating cAMP homeostasis, dysfunctional PDE ...

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Real-time Fluorescence Measurement of Enterovirus Uncoating Authors: Visa Ruokolainen, Mira Laajala and Varpu Marjomäki, date: 04/05/2020, view: 1593, Q&A: 0

Viruses need to open, i.e., uncoat, in order to release their genomes for efficient replication and translation. Especially for non-enveloped viruses, such as enteroviruses, the cues leading to uncoating are less well known. The status of the virus has previously been observed mainly by transmission electron microscopy using negative ...

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Neuroscience

A Simple and Fast Battery Test for Phenotypic Characterization of Mice Authors: Mariana S. A. Garcia-Gomes, Dennis A. Zanatto, Pedro K. Yamamoto, Danilo Wadt, Ana T. F. B. Antiorio, Jilma Aleman-Laporte, Sandra R. Alexandre-Ribeiro, Guilherme A. Marson, Cezar Guizzo, Silvia M. G. Massironi, Maria M. Bernardi and Claudia M. C. Mori, date: 04/05/2020, view: 1798, Q&A: 0

Despite the great number of test batteries already known to assess the behavior of genetically modified and inbred strains of mice, only a few of them focus on basic neurological parameters. The purpose of the battery test proposed is to settle a specific methodology to characterize the phenotype of neurological disease models in mutant or ...

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Past Issues in 2020

Volume: 10 Issue: 7

Biochemistry

Cancer Biology

Cell Biology

Developmental Biology

Microbiology

Molecular Biology

Neuroscience