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    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: 527, 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: 803, 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: 640, 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: 1595, 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 ...
    A General Method for Intracellular Protein Delivery through ‘E-tag’ Protein Engineering and Arginine Functionalized Gold Nanoparticles
    Authors:  Rubul Mout and Vincent M. Rotello, date: 12/20/2017, view: 1212, Q&A: 0
    [Abstract] In this protocol, we describe a method for direct cytosolic protein delivery that avoids endosomal entrapment of the delivered proteins. We achieved this by tagging the desired protein with an oligo glutamic acid tag (E-tag), and subsequently using carrier gold nanoparticles to deliver these E-tagged proteins. When E-tagged proteins and ...
    Measuring the Endocytic Recycling of Amyloid Precursor Protein (APP) in Neuro2a Cells
    Authors:  Florent Ubelmann, Tatiana Burrinha and Claudia Guimas Almeida, date: 12/05/2017, view: 1225, Q&A: 0
    [Abstract] The established primary trigger of Alzheimer’s disease’s is β-amyloid (Aβ) (Mucke and Selkoe, 2012). Amyloid precursor protein (APP) endocytosis is required for Aβ generation at early endosomes (Rajendran and Annaert, 2012). APP retention at endosomes also depends on its recycling back to the plasma membrane (Koo et al., 1996; Ubelmann et ...
    Bioluminescence Resonance Energy Transfer (BRET) Assay for Determination of Molecular Interactions in Living Cells
    [Abstract] The bioluminescence resonance energy transfer (BRET) assay can be used as an indicator of molecular approximation and/or interaction. A significant resonance energy transfer signal is generated when the acceptor, having the appropriate spectral overlap with the donor emission, is approximated with the donor. In the example provided, proteins ...
    Detection of Membrane Protein Interactions by Cell-based Tango Assays
    [Abstract] The Tango assay is a protein-protein interaction assay, in which a transcription factor (rTA) is fused to a membrane-bound protein via a linker that contains a cleavage site for TEV protease, whereas a soluble interaction partner is fused to TEV protease (Barnea et al., 2008). Association between the two interaction partners leads to an ...
    Streptavidin Bead Pulldown Assay to Determine Protein Homooligomerization
    [Abstract] Pulldown assay is a conventional method to determine protein-protein interactions in vitro. Expressing a protein of interest with two different tags allows testing whether both versions can be captured via one of the two tags as homooligomeric complex. This protocol is based on streptavidin bead capture of a biotinylated protein and ...
    γ-Secretase Epsilon-cleavage Assay
    [Abstract] γ-Secretase epsilon-cleavage assay is derived from the cell-based Tango assay (Kang et al., 2015), and is a fast and sensitive method to determine the initial cleavage of C99 by γ-secretase. In this protocol, we use HTL cells, which are HEK293 cells with a stably integrated luciferase reporter under the control of the bacterial tetO ...