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Past Issues in 2019

Volume: 9 Issue: 11

Biochemistry

Glucosidase Inhibition to Study Calnexin-assisted Glycoprotein Folding in Cells Authors: Hao Wang and Qingyu Wu, date: 06/05/2019, view: 3835, Q&A: 0

Calnexin is a chaperone protein that plays a critical role in glycoprotein folding in the endoplasmic reticulum (ER). The function of calnexin depends on its binding to monoglucosylated oligosaccharides on nascent glycoproteins, whereas the generation of monoglucosylated oligosaccharides depends on the activity of α-glucosidases I and II, which ...

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Tryptophan Fluorescence Quenching Assays for Measuring Protein-ligand Binding Affinities: Principles and a Practical Guide Authors: Anthony Yammine, Jinlong Gao and Ann H. Kwan, date: 06/05/2019, view: 16176, Q&A: 0

Tryptophan fluorescence quenching is a type of fluorescence spectroscopy used for binding assays. The assay relies on the ability to quench the intrinsic fluorescence of tryptophan residues within a protein that results from changes in the local environment polarity experienced by the tryptophan(s) upon the addition of a binding partner or ligand. ...

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Cross-linking, Immunoprecipitation and Proteomic Analysis to Identify Interacting Proteins in Cultured Cells Authors: Hao Wang, Meiling He, Belinda Willard and Qingyu Wu, date: 06/05/2019, view: 11256, Q&A: 0

Extracellular expression is essential for the function of secreted and cell surface proteins. Proper intracellular trafficking depends on protein interactions in multiple subcellular compartments. Co-immunoprecipitation and the yeast two-hybrid system are commonly used to investigate protein-protein interactions. These methods, however, depend on ...

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A Detailed Protocol for Large-scale Recombinant Expression and Validation of Human FGFR2 with Its Transmembrane and Extracellular Domains in Escherichia coli Authors: Adam Bajinting and Ho Leung Ng, date: 06/05/2019, view: 4317, Q&A: 0

Receptor tyrosine kinases (RTKs) are an important class of transmembrane receptors that mediate some of the most crucial biochemical pathways essential to the growth, differentiation, and survival of a cell and thus, are highly involved in cancers. Due to the complexity of RTKs having biochemically different domains including a transmembrane ...

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Immunology

Detection of Antigen-specific T cells in Spleens of Vaccinated Mice Applying ³[H]-Thymidine Incorporation Assay and Luminex Multiple Cytokine Analysis Technology Authors: Maria Agallou and Evdokia Karagouni, date: 06/05/2019, view: 9379, Q&A: 0

For many infectious diseases T cells are an important part of naturally acquired protective immune responses, and inducing these by vaccination has been the aim of much research. Here, we describe a protocol for the analysis of vaccine-induced antigen-specific immune responses. For this purpose, cells of whole spleens obtained from vaccinated ...

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Measurement of CD74 N-terminal Fragment Accumulation in Cells Treated with SPPL2a Inhibitor Authors: Rubén Martínez-Barricarte, Xiao-Fei Kong and Jean-Laurent Casanova, date: 06/05/2019, view: 4480, Q&A: 0

The recent discovery of human signal peptide peptidase-like 2a (SPPL2a) deficiency in humans revealed the toxicity associated with the accumulation of one of its substrates, CD74 N-terminal fragment (CD74-NTF), for certain type of dendritic cells (cDC2). We developed a two-step protocol for monitoring the accumulation of this molecule in different ...

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Molecular Biology

Improved Mammalian Mitochondrial RNA Isolation Authors: Jinliang Huang and Geng Wang, date: 06/05/2019, view: 5863, Q&A: 0

Mitochondria have two sets of RNAs. One is encoded in mitochondrial genome, and the other that consists of imported RNAs within mitochondria and cytosolic RNAs associated with mitochondrial outer membrane is encoded in the nucleus. These mitochondrial RNAs play important roles in mitochondrion biosynthesis and signaling in and out of mitochondria. ...

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Minigene Assay to Evaluate CRISPR/Cas9-based Excision of Intronic Mutations that Cause Aberrant Splicing in Human Cells Authors: David J. Sanz and Patrick T. Harrison, date: 06/05/2019, view: 4917, Q&A: 0

The construction of Hybrid minigenes provides a robust and simple strategy to study the effects of disease-causing mutations on mRNA splicing when biological material from patient cells is not available. Hybrid minigenes can be used as splicing reporter plasmids allow RNA expression and heterologous splicing reactions between synthetic splicing ...

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Organelle-associated rRNA Degradation Authors: Jinliang Huang and Geng Wang, date: 06/05/2019, view: 4275, Q&A: 0

Cytosolic rRNAs are highly dynamic and can be degraded under conditions such as apoptosis, starvation and magnesium depletion. The degradation is also related to their specific localization, as fractions of cytosolic ribosomes are localized on the surfaces of intracellular organelles, such as endoplasmic reticulum (ER) and mitochondria. Such ...

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Neuroscience

Accessing Olfactory Habituation in Drosophila melanogaster with a T-maze Paradigm Authors: Ourania Semelidou, Summer F. Acevedo and Efthimios M.C. Skoulakis, date: 06/05/2019, view: 5192, Q&A: 0

Habituation is the process whereby perceptual changes alter the value of environmental stimuli, enabling salience filtering. This behavioral response decrement is a form of non-associative learning, where the subject learns about the stimulus and does not involve sensory adaptation, sensory or motor fatigue. The range of behavioral responses in D. ...

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Plant Science

Transient Expression Assay in Strawberry Fruits Authors: Mengting Pi, Qi Gao and Chunying Kang, date: 06/05/2019, view: 5984, Q&A: 0

Strawberry, including the woodland strawberry Fragaria vesca (2x) and the cultivated strawberry (Fragaria × ananassa, 8x), has emerged as a model system for studying fruit development and ripening. Transient expression provides a quick assay for gene functions or gene interactions. In strawberry, ...

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Simple Method to Determine Protein Redox State in Arabidopsis thaliana Authors: Keisuke Yoshida and Toru Hisabori, date: 06/05/2019, view: 5568, Q&A: 1

Thiol-based redox regulation is a posttranslational protein modification that plays a key role in many biological aspects. To understand its regulatory functions, we need a method to directly assess protein redox state in vivo. Here we present a simple procedure to determine protein redox state in a model plant Arabidopsis thaliana ...

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