Mammalia

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    Protocols in Current Issue
    Single-cell qPCR Assay with Massively Parallel Microfluidic System
    Authors:  Marta Prieto-Vila, Takahiro Ochiya and Yusuke Yamamoto, date: 03/20/2020, view: 183, Q&A: 0
    [Abstract] The single-cell transcriptome is the set of messenger RNA molecules expressed in one cell. It is extremely variable and changes according to external, physical and biochemical conditions. Due to sensitivity shortages, most of genetic studies use bulk samples, providing only the average gene expression. Single-cell technologies have provided a ...
    Cell-free Reconstitution of the Packaging of Cargo Proteins into Vesicles at the trans Golgi Network
    Authors:  Xiao Tang, Feng Yang and Yusong Guo, date: 03/05/2020, view: 301, Q&A: 0
    [Abstract] Protein sorting at the trans Golgi network (TGN) plays important roles in targeting newly synthesized proteins to their specific destinations. The aim of this proposal is to reconstitute the packaging of non-Golgi resident cargo proteins into vesicles at the TGN, utilizing rat liver cytosol, semi-intact mammalian cells and nucleotides. ...
    Estimating Cellular Abundances of Halo-tagged Proteins in Live Mammalian Cells by Flow Cytometry
    Authors:  Claudia Cattoglio, Xavier Darzacq, Robert Tjian and Anders Sejr Hansen, date: 02/20/2020, view: 713, Q&A: 0
    [Abstract] Accurate abundance measurements of cellular proteins are required to achieve a quantitative and predictive understanding of any biological process inside the cell. Existing methods to determine absolute protein abundances are labor-intensive and/or require sophisticated experimental and computational infrastructure (e.g., fluorescence ...
    ChIP-Seq from Limited Starting Material of K562 Cells and Drosophila Neuroblasts Using Tagmentation Assisted Fragmentation Approach
    Authors:  Junaid Akhtar, Piyush More and Steffen Albrecht, date: 02/20/2020, view: 258, Q&A: 0
    [Abstract] Chromatin immunoprecipitation is extensively used to investigate the epigenetic profile and transcription factor binding sites in the genome. However, when the starting material is limited, the conventional ChIP-Seq approach cannot be implemented. This protocol describes a method that can be used to generate the chromatin profiles from as low as ...
    Detection of Individual RNA in Fixed Cells and Tissues by Chromogenic ISH
    Authors:  Meng Jiang, Chen Lin and Rongqin Ke, date: 02/05/2020, view: 324, Q&A: 0
    [Abstract] Visualization of RNA molecules in situ helps to better understand the functions of expressed genes. Currently, most conventional in situ hybridization methods for visualization of individual RNAs are based on fluorescence detection. Herein we present a chromogenic in situ hybridization protocol for visualization of ...
    Identification of Target Protein for Bio-active Small Molecule Using Photo-cross Linked Beads and MALDI-TOF Mass Spectrometry
    [Abstract] Development of methods for protein identification is one of the important aspects of proteomics. Here, we report a protocol for the preparation of compound conjugated beads by photo-crosslinking, affinity purification, gel electrophoresis, and highly sensitive mass spectrometric assay for drug-target identification. Although there are several ...
    Approaching RNA-seq for Cell Line Identification
    Authors:  Tabrez A. Mohammad and Yidong Chen, date: 02/05/2020, view: 502, Q&A: 0
    [Abstract] Cancer cell lines serve as invaluable model systems for cancer biology research and help in evaluating the efficacy of new therapeutic agents. However, cell line contamination and misidentification have become one of the most pressing problems affecting biomedical research. Available methods of cell line authentication suffer from limited access, ...
    Live Mitochondrial or Cytosolic Calcium Imaging Using Genetically-encoded Cameleon Indicator in Mammalian Cells
    Authors:  Elisa Greotti and Tullio Pozzan, date: 02/05/2020, view: 429, Q&A: 0
    [Abstract] Calcium (Ca2+) imaging aims at investigating the dynamic changes in live cells of its concentration ([Ca2+]) in different pathophysiological conditions. Ca2+ is an ubiquitous and versatile intracellular signal that modulates a large variety of cellular functions thanks to a cell type-specific toolkit and a complex ...
    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: 673, Q&A: 0
    [Abstract] 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 ...
    Ribonucleoprotein Immunoprecipitation (RIP) Analysis
    Authors:  Jennifer L. Martindale, Myriam Gorospe and Maria L. Idda, date: 01/20/2020, view: 592, Q&A: 0
    [Abstract] 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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