Protocols in Current Issue
    Integrin Dependent RhoB Activation Assay Using Leukocytes
    Authors:  Janne Lehtonen, Katarzyna Potrzebowska, Malin Samuelsson and Lena Svensson, date: 12/05/2018, view: 642, Q&A: 0
    [Abstract] To be able to migrate, leukocyte needs to re-use its adhesion molecules to move forward. These adhesion molecules are called integrins and are intracellularly transported via endocytosis and exocytosis in order to translocate to a new site on the cell membrane. The intracellular transportation is regulated by different small GTPases including ...
    Wheat Germ Agglutinin (WGA)-SDS-PAGE: A Novel Method for the Detection of O-GlcNAc-modified Proteins by Lectin Affinity Gel Electrophoresis
    Authors:  Ko Fujioka, Yuji Kubota and Mutsuhiro Takekawa, date: 12/05/2018, view: 1218, Q&A: 0
    [Abstract] Diverse cytoplasmic and nuclear proteins dynamically change their molecular functions by O-linked β-N-acetylglucosamine (O-GlcNAc) modification on serine and/or threonine residues. Evaluation of the O-GlcNAcylation level of a specific protein, however, needs multiple and time-consuming steps if using conventional methods (e.g., ...
    A Functionally Robust Phenotypic Screen that Identifies Drug Resistance-associated Genes Using 3D Cell Culture
    Authors:  Sun-Young Lee and Mina J. Bissell, date: 11/20/2018, view: 1384, Q&A: 0
    [Abstract] Drug resistance is a major obstacle in cancer treatment: A case in point is the failure of cancer patients to respond to tyrosine kinase inhibitors (TKI) of EGFR, a receptor that is highly expressed in many cancers. Identification of the targets and delineation of mechanisms of drug resistance remain major challenges. Traditional pharmacological ...
    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: 1129, 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 ...
    Detection of Internal Matrix Targeting Signal-like Sequences (iMTS-Ls) in Mitochondrial Precursor Proteins Using the TargetP Prediction Tool
    Authors:  Felix Boos, Timo Mühlhaus and Johannes M. Herrmann, date: 09/05/2018, view: 1307, Q&A: 0
    [Abstract] Mitochondria contain hundreds of proteins which are encoded by the nuclear genome and synthesized in the cytosol from where they are imported into the organelle. Sorting signals encoded in the primary and secondary sequence of these proteins mediate the recognition of newly synthesized precursor proteins and their subsequent translocation through ...
    Deoxycholate Fractionation of Fibronectin (FN) and Biotinylation Assay to Measure Recycled FN Fibrils in Epithelial Cells
    Authors:  Archana Varadaraj, Carina Magdaleno and Karthikeyan Mythreye, date: 08/20/2018, view: 1263, Q&A: 0
    [Abstract] Fibronectin (FN) is an extracellular matrix protein that is secreted by many cell types and binds predominantly to the cell surface receptor Integrin α5β1. Integrin α5β1 binding initiates the step-wise assembly of FN into fibrils, a process called fibrillogenesis. We and several others have demonstrated critical effects of fibrillogenesis on cell ...
    Mammalian Cell-derived Vesicles for the Isolation of Organelle Specific Transmembrane Proteins to Conduct Single Molecule Studies
    Authors:  Faruk H. Moonschi, Ashley M. Fox-Loe, Xu Fu and Chris I. Richards, date: 05/05/2018, view: 2000, Q&A: 0
    [Abstract] Cell-derived vesicles facilitate the isolation of transmembrane proteins in their physiological membrane maintaining their structural and functional integrity. These vesicles can be generated from different cellular organelles producing, housing, or transporting the proteins. Combined with single-molecule imaging, isolated organelle specific ...
    The Long-lived Protein Degradation Assay: an Efficient Method for Quantitative Determination of the Autophagic Flux of Endogenous Proteins in Adherent Cell Lines
    Authors:  Morten Luhr, Frank Sætre and Nikolai Engedal, date: 05/05/2018, view: 3029, Q&A: 0
    [Abstract] Autophagy is a key player in the maintenance of cellular homeostasis in eukaryotes, and numerous diseases, including cancer and neurodegenerative disorders, are associated with alterations in autophagy. The interest for studying autophagy has grown intensely in the last two decades, and so has the arsenal of methods utilised to study this highly ...
    A Method for Extracting the Nuclear Scaffold from the Chromatin Network
    Authors:  Junjie Chen, Boon Heng Dennis Teo and Jinhua Lu, date: 04/20/2018, view: 2048, Q&A: 0
    [Abstract] Each cell contains many large DNA polymers packed in a nucleus of approx. 10 μm in diameter. With histones, these DNA polymers are known to form chromatins. How chromatins further compact in the nucleus is unclear but it inevitably depends on an extensive non-chromatin nuclear scaffold. Imaging of endogenous chromatin network and the complementary ...
    Visible Immunoprecipitation (VIP) Assay: a Simple and Versatile Method for Visual Detection of Protein-protein Interactions
    Authors:  Yohei Katoh, Kentaro Nakamura and Kazuhisa Nakayama, date: 01/05/2018, view: 3991, Q&A: 0
    [Abstract] The visible immunoprecipitation (VIP) assay is a convenient alternative to conventional co-immunoprecipitation (Katoh et al., 2015). By processing lysates from cells co-expressing GFP-fusion and RFP-fusion proteins for immunoprecipitation with GST-tagged anti-GFP Nanobody and glutathione-Sepharose beads, protein-protein interactions can ...

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