Mammalia

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    Protocols in Current Issue
    Production and Purification of Dengue Virus-like Particles from COS-1 Cells
    Authors:  Jedhan Ucat Galula, Gwong-Jen J. Chang and Day-Yu Chao, date: 06/20/2019, view: 916, Q&A: 0
    [Abstract] Non-infectious virus-like particles (VLPs) containing dengue virus (DENV) pre-membrane (prM) and envelope (E) proteins have been demonstrated to be highly immunogenic and can be used as a potential vaccine candidate as well as a tool for serodiagnostic assays. Successful application of VLPs requires abundant, and high-purity production methods. ...
    DNA Immunization Using in vivo Electroporation for Generating Monoclonal Antibodies Against Mouse IL-9R
    [Abstract] Membrane proteins such as cytokine receptors and G protein-coupled receptors can be drug targets. Recently, we have generated specific monoclonal antibodies (mAbs) against the mouse IL-9 receptor (IL-9R) and found that IL-9R on memory B cells have critical roles in T-dependent immune response. So far, most antibodies against cell surface proteins ...
    Electroporation of Labeled Antibodies to Visualize Endogenous Proteins and Posttranslational Modifications in Living Metazoan Cell Types
    Authors:  Sascha Conic, Dominique Desplancq, László Tora and Etienne Weiss, date: 11/05/2018, view: 1671, Q&A: 0
    [Abstract] The spatiotemporal localization of different intracellular factors in real-time and their detection in live cells are important parameters to understand dynamic protein-based processes. Therefore, there is a demand to perform live-cell imaging and to measure endogenous protein dynamics in single cells. However, fluorescent labeling of endogenous ...
    Structural Analysis of Target Protein by Substituted Cysteine Accessibility Method
    Authors:  Tetsuo Cai and Taisuke Tomita, date: 09/05/2018, view: 1872, Q&A: 0
    [Abstract] Substituted Cysteine Accessibility Method (SCAM) is a biochemical approach to investigate the water accessibility or the spatial distance of particular cysteine residues substituted in the target protein. Protein topology and structure can be annotated by labeling with methanethiosulfonate reagents that specifically react with the cysteine ...
    Fluorometric Estimation of Glutathione in Cultured Microglial Cell Lysate
    Authors:  Vikas Singh, Ruchi Gera, Mahaveer Prasad Purohit, Satyakam Patnaik and Debabrata Ghosh, date: 06/05/2017, view: 5944, Q&A: 5
    [Abstract] Glutathione is one of the major antioxidant defense components present in cells. It is predominantly present as reduced glutathione (GSH) and converted into oxidized glutathione (GSSG) while reducing the free radicals like hydroxyl ions (OH-). For the measurement of GSH and GSSG, o-phthalaldehyde (OPT) has been used as a fluorescent ...
    Measuring Procaspase-8 and -10 Processing upon Apoptosis Induction
    Authors:  Sabine Pietkiewicz, Clara Wolfe, Jörn H. Buchbinder and Inna N. Lavrik, date: 01/05/2017, view: 5329, Q&A: 0
    [Abstract] Apoptosis or programmed cell death is important for multicellular organisms to keep cell homeostasis and for the clearance of mutated or infected cells. Apoptosis can be induced by intrinsic or extrinsic stimuli. The first event in extrinsic apoptosis is the formation of the Death-Inducing Signalling Complex (DISC), where the initiator caspases-8 ...
    Activity-based Pull-down of Proteolytic Standard and Immunoproteasome Subunits
    [Abstract] Activity-based probes (ABP) are small organic molecules that irreversibly bind to the active center of a specific enzyme family and may be coupled to a fluorophore or an affinity tag (Li et al., 2013). Here, we describe a method to pull-down active catalytic standard and immunoproteasome subunits in cell lysates using the biotinylated, ...
    Detection of Wnt5 in Media Conditioned by Mouse Embryonic Fibroblast
    Authors:  Juan Flores and Nan Gao, date: 10/20/2016, view: 4831, Q&A: 0
    [Abstract] This protocol describes the procedure of visualizing secreted Wnt5 protein in serum free media via western blotting. This procedure can also be used to visualize other secreted proteins larger than 10,000 daltons. The work presented in this paper visualizes Wnt5 secreted by mouse embryonic fibroblast (MEF), but can be adapted to other cell lines ...
    Spectrophotometric Determination of Glutamine Synthetase Activity in Cultured Cells
    Authors:  I-Chen Peng, Alex J. Bott and Wei-Xing Zong, date: 10/05/2016, view: 7384, Q&A: 0
    [Abstract] Glutamine synthetase (GS), which catalyzes the conversion of glutamate and ammonia to glutamine, is widely distributed in animal tissues and cell culture lines. The importance of this enzyme is suggested by the fact that glutamine, the product of GS-catalyzed de novo synthesis reaction, is the most abundant free amino acid in blood (Smith and ...
    Identification of RNA-binding Proteins
    Authors:  Kazuya Masuda and Tadamitsu Kishimoto, date: 09/05/2016, view: 7896, Q&A: 0
    [Abstract] This protocol describes the extraction of RNA-binding proteins (RBPs) from cell lysates. In order to pull down target RBPs, 5-bromo-UTP (BrUTP)-incorporated RNA probes are used, which are generated by in vitro transcription. The schematic diagram (Flowchart) with procedure is indicated (Figure1 and Figure 2).

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