Protocols in Current Issue
    Measuring Protein Synthesis during Cell Cycle by Azidohomoalanine (AHA) Labeling and Flow Cytometric Analysis
    Authors:  Koshi Imami and Tomoharu Yasuda, date: 04/20/2019, view: 721, Q&A: 0
    [Abstract] Protein synthesis is one of the most fundamental biological processes to maintain cellular proteostasis. Azidohomoalaine (AHA) is a non-radioactive and “clickable” amino acid analog of methionine which can be incorporated into newly synthesized proteins. Thus, AHA-labeled nascent proteins can be detected and quantified through fluorescent labeling ...
    Quantitative Analysis of Cargo Density in Single-extracellular Vesicles by Imaging
    Authors:  Taketoshi Kajimoto and Shun-ichi Nakamura, date: 12/20/2018, view: 1148, Q&A: 0
    [Abstract] Function of extracellular vesicles such as exosomes and microvesicles is determined by their wide ranges of cargoes inside them. Even in the pure exosomes or microvesicles the cargo contents are very heterogeneous. To understand this heterogeneous nature of extracellular vesicles, we need information of the vesicles, which will give us some ...
    Electron Microscopic Detection of Proteins and Protein Complexes in Mammalian Cells Using APEX-tagged, Conditionally Stable Nanobodies
    Authors:  Thomas E. Hall, James Rae, Nicholas Ariotti and Robert G. Parton, date: 11/20/2018, view: 1182, Q&A: 0
    [Abstract] The recent development of 3D electron microscopic techniques for cells and tissues has necessitated the development of new methods for the detection of proteins and protein-complexes in situ. The development of new genetic tags, such as the ascorbate peroxidase, APEX, for electron microscopic detection of tagged proteins has expanded the ...
    Ciliary Assembly/Disassembly Assay in Non-transformed Cell Lines
    Authors:  Masaki Saito, Kensuke Sakaji, Wataru Otsu and Ching-Hwa Sung, date: 03/20/2018, view: 3106, Q&A: 0
    [Abstract] The primary cilium is a non-motile sensory organelle whose assembly and disassembly are closely associated with cell cycle progression. The primary cilium is elongated from the basal body in quiescent cells and is resorbed as the cells re-enter the cell cycle. Dysregulation of ciliary dynamics has been linked with ciliopathies and other human ...
    Notch Ligand Binding Assay Using Flow Cytometry
    Authors:  Shweta Varshney and Pamela Stanley, date: 12/05/2017, view: 4342, Q&A: 0
    [Abstract] Notch signaling is an evolutionarily conserved signaling pathway that plays an indispensable role during development, and in the maintenance of homeostatic processes, in a wide variety of tissues (Kopan, 2012; Hori et al., 2013). The multifaceted roles of Notch signaling are stringently regulated at different levels. One of the most ...
    Peroxisome Motility Measurement and Quantification Assay
    Authors:  Jeremy Metz, Inês G. Castro and Michael Schrader, date: 09/05/2017, view: 3243, Q&A: 0
    [Abstract] Organelle movement, distribution and interaction contribute to the organisation of the eukaryotic cell. Peroxisomes are multifunctional organelles which contribute to cellular lipid metabolism and ROS homeostasis. They distribute uniformly in mammalian cells and move along microtubules via kinesin and dynein motors. Their metabolic cooperation ...
    Advanced Design of Minimalistic Dumbbell-shaped Gene Expression Vectors
    Authors:  Xiaoou Jiang and Volker Patzel, date: 08/05/2017, view: 3984, Q&A: 0
    [Abstract] Minimal DNA vectors exclusively comprising therapeutically relevant sequences hold great promise for the development of novel therapeutic regimen. Dumbbell-shaped vectors represent non-viral non-integrating DNA minimal vectors which have entered an advanced stage of clinical development (Hardee et al., 2017). Spliceable introns and DNA ...
    Measurement of the Intracellular Calcium Concentration with Fura-2 AM Using a Fluorescence Plate Reader
    Authors:  Magdiel Martínez, Namyr A. Martínez and Walter I. Silva, date: 07/20/2017, view: 12900, Q&A: 0
    [Abstract] Intracellular calcium elevation triggers a wide range of cellular responses. Calcium responses can be affected or modulated by membrane receptors mutations, localization, exposure to agonists/antagonists, among others (Burgos et al., 2007; Martínez et al., 2016). Changes in intracellular calcium concentration can be measured ...
    Generation of a Cellular Reporter for Functional BRD4 Inhibition
    Authors:  Sara Sdelci and Stefan Kubicek, date: 07/05/2017, view: 3038, Q&A: 0
    [Abstract] The ubiquitously expressed bromodomain-containing protein 4 (BRD4) is an epigenetic reader, which recruits transcriptional regulatory complexes to acetylated chromatin. Because of its role in enhancing proliferation, BRD4 has become a therapeutic target in oncology, as the inhibition of this protein leads to the reduction of the growth of many ...
    Functional Analysis of Connexin Channels in Cultured Cells by Neurobiotin Injection and Visualization
    Authors:  Philipp Wörsdörfer and Klaus Willecke, date: 06/05/2017, view: 4126, Q&A: 0
    [Abstract] Functional gap junction channels between neighboring cells can be assessed by microinjection of low molecular weight tracer substances into cultured cells. The extent of direct intercellular communication can be precisely quantified by this method. This protocol describes the iontophoretic injection and visualisation of Neurobiotin into cultured ...

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