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    MicroScale Thermophoresis as a Tool to Study Protein-peptide Interactions in the Context of Large Eukaryotic Protein Complexes
    Authors:  Maximilian G. Plach, Klaus Grasser and Thomas Schubert, date: 12/05/2017, view: 589, Q&A: 0
    [Abstract] Protein-peptide interactions are part of many physiological processes, for example, epigenetics where peptide regions of histone complexes are crucial for regulation of chromatin structure. Short peptides are often also used as alternatives to small molecule drugs to target protein complexes. Studying the interactions between proteins and peptides ...
    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 ...
    Preparation of Crude Synaptosomal Fractions from Mouse Brains and Spinal Cords
    Author:  Oliver Wirths, date: 08/05/2017, view: 1322, Q&A: 0
    [Abstract] The current protocol describes the preparation of crude synaptosomal fractions from mouse brain or spinal cord samples. In detail, a sequential protocol yielding crude synaptosomal and light membrane fractions is provided. This fast and easy method might be sufficient to assess the amount of synaptic proteins in down-steam applications like ...
    Biochemical Analysis of Caspase-8-dependent Proteolysis of IRF3 in Virus-infected Cells
    Authors:  Gayatri Subramanian, Karen Pan, Ritu Chakravarti and Saurabh Chattopadhyay, date: 11/20/2016, view: 2417, Q&A: 0
    [Abstract] Interferon regulatory factor 3 (IRF3) is a transcription factor, which is critical for the antiviral response against a wide range of viruses (Hiscott, 2007; Ikushima et al., 2013). It gets activated in virus-infected cells via Toll like receptors (TLRs), RIG-I (retinoic acid inducible gene 1) like receptors (RLRs), cyclic GMP-AMP ...
    Generation of IgG-Fc Glycovariants Using Recombinant Glycosidases and Glycosyltransferases
    Authors:  Isaak Quast, Michael A. Maurer and Jan D. Lünemann, date: 08/05/2016, view: 2997, Q&A: 0
    [Abstract] The immunoglobulin G (IgG) fragment crystallizable (Fc) domain contains a single, highly conserved asparagine 297 (N297) glycosylation site in the CH2 domain, which is buried within the hydrophobic core of each of the two heavy chains. The biantennary core glycan structure, composed of 2 N-acetylglucosamine (GlcNAc) and 3 mannose residues, can be ...
    An in vitro Transcription/translation System for Detection of Protein Interaction
    Authors:  Pin-Chun Lin, Ya-Chun Chang and Shih-Shun Lin, date: 05/05/2016, view: 4336, Q&A: 0
    [Abstract] Studying protein-protein interaction is crucial to understand the fundamental processes of molecular biology. High-throughput screening, such as immunoprecipitation followed by proteomic analysis, allows for the identification of numerous candidate partners that might interact with a selected protein. However, experimental validation of ...
    Actin Retrograde Flow in Permeabilized Cells: Myosin-II Driven Centripetal Movement of Transverse Arcs
    Authors:  Yee Han Tee and Alexander D. Bershadsky, date: 03/05/2016, view: 2533, Q&A: 0
    [Abstract] Numerous biological functions such as cytokinesis, changes in cell shape and cell migration require actomyosin-driven cellular contractility. However, the detailed mechanism of how contractile forces drive cellular processes are difficult to decipher due to the complexity of the intracellular environment. In particular, the mesoscopic description ...