Mammalia

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    Protocols in Current Issue
    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: 146, Q&A: 0
    [Abstract] 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 ...
    Glucosidase Inhibition to Study Calnexin-assisted Glycoprotein Folding in Cells
    Authors:  Hao Wang and Qingyu Wu, date: 06/05/2019, view: 140, Q&A: 0
    [Abstract] 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 ...
    Organelle-associated rRNA Degradation
    Authors:  Jinliang Huang and Geng Wang, date: 06/05/2019, view: 111, Q&A: 0
    [Abstract] 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 ...
    Improved Mammalian Mitochondrial RNA Isolation
    Authors:  Jinliang Huang and Geng Wang, date: 06/05/2019, view: 127, Q&A: 0
    [Abstract] 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. ...
    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: 118, Q&A: 0
    [Abstract] 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 ...
    A Flow Cytometric Method to Determine Transfection Efficiency
    [Abstract] Mammalian cell transfection is a powerful technique commonly used in molecular biology to express exogenous DNA or RNA in cells and study gene and protein function. Although several transfection strategies have been developed, there is a wide variation with regards to transfection efficiency, cell toxicity and reproducibility. Thus, a sensitive ...
    Triple Fluorescence Anisotropy Reporter Imaging in Living Cells
    Authors:  Brian Ross, Shenq Huey Wong, Nicole E. Snell, Jin Zhang and M. A. Rizzo, date: 05/05/2019, view: 445, Q&A: 0
    [Abstract] FRET-based genetically encoded biosensors incorporate two fluorescent proteins into their design to enable ratiometric biosensing of signaling activities in live cells. While emission ratios are generally useful for quantitative studies, they leave little room in the optical spectrum for additional sensors and optogenetic tools. Homotransfer-based ...
    Northern Blot with IR Fluorescent Probes: Strategies for Probe Preparation
    Authors:  Christopher Fields, Peike Sheng, Bret Miller, Tianqi Wei and Mingyi Xie, date: 04/20/2019, view: 605, Q&A: 0
    [Abstract] Northern blot is a molecular biology technique that can detect, quantify, and determine the molecular weight of RNA. Recently, we published a protocol utilizing near-infrared (IR) fluorescent probes in Northern blot (irNorthern). Our method is as sensitive as other non-radioactive methods but is more straightforward and versatile. Additionally, we ...
    Identification of RNase-sensitive LINE-1 Ribonucleoprotein Interactions by Differential Affinity Immobilization
    Authors:  Hua Jiang, Martin S. Taylor, Kelly R. Molloy, Ilya Altukhov and John LaCava, date: 04/05/2019, view: 716, Q&A: 0
    [Abstract] Long Interspersed Nuclear Element-1 (LINE-1, L1) constitutes a family of autonomous, self-replicating genetic elements known as retrotransposons. Although most are inactive, copious L1 sequences populate the human genome. L1s proliferate in a ‘copy-and-paste’ fashion through an RNA intermediate; a full-length L1 transcript is ~6,000 nucleotides ...
    Quantification of Hepatitis B Virus Covalently Closed Circular DNA in Infected Cell Culture Models by Quantitative PCR
    Authors:  Bingqian Qu and Stephan Urban, date: 04/05/2019, view: 584, Q&A: 0
    [Abstract] Persistence of the human hepatitis B virus (HBV) requires the maintenance of covalently closed circular (ccc)DNA, the episomal genome reservoir in nuclei of infected hepatocytes. cccDNA elimination is a major aim in future curative therapies currently under development. In cell culture based in vitro studies, both hybridization- and ...



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