Molecular Biology

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    Protocols in Current Issue
    Generation and Testing of Fluorescent Adaptable Simple Theranostic (FAST) Proteins
    [Abstract] This protocol provides a step-by-step method to create recombinant fluorescent fusion proteins that can be secreted from mammalian cell lines. This builds on many other recombinant protein and fluorescent protein techniques, but is among the first to harness fluorescent fusion proteins secreted directly into cell culture supernatant. This opens ...
    In vitro Assessment of Pathogen Effector Binding to Host Proteins by Surface Plasmon Resonance
    [Abstract] The mechanisms of virulence and immunity are often governed by molecular interactions between pathogens and host proteins. The study of these interactions has major implications on understanding virulence activities, and how the host immune system recognizes the presence of pathogens to initiate an immune response. Frequently, the association ...
    Molecular Size Analysis of Recombinant Importin-histone Complexes Using Analytical Ultracentrifugation
    Authors:  Abhilash Padavannil, Chad A. Brautigam and Yuh Min Chook, date: 05/20/2020, view: 616, Q&A: 0
    [Abstract] Histones constitute the protein components of nucleosomes. Despite their small sizes, histones do not diffuse through the nuclear pore complex. Instead, they are transported to the nucleus by importins, either alone or in complex with histone chaperones. Determining the molecular size of the importin-histone complexes is key to understanding the ...
    Generation, Analyzing and in-vivo Drug Treatment of Drosophila Models with IBMPFD
    Authors:  Ting Zhang, Bruce A. Hay and Ming Guo, date: 05/20/2020, view: 576, Q&A: 0
    [Abstract] Missense mutations of p97/cdc48/Valosin-containing protein (VCP) cause inclusion body myopathy, Paget disease with frontotemporal dementia (IBMPFD) and other neurodegenerative diseases. The pathological mechanism of IBMPFD is not clear and there is no treatment. We generated Drosophila models of IBMPFD in adult flight muscle in ...
    Split Nano Luciferase-based Assay to Measure Assembly of Japanese Encephalitis Virus
    Authors:  Simon Goto, Kotaro Ishida, Ryosuke Suzuki and Eiji Morita, date: 05/05/2020, view: 459, Q&A: 0
    [Abstract] Cells infected with flavivirus release various forms of infectious and non-infectious particles as products and by-products. Comprehensive profiling of the released particles by density gradient centrifugation is informative for understanding viral particle assembly. However, it is difficult to detect low-abundance minor particles in such ...
    Bimolecular Fluorescence Complementation (BiFC) for Studying Sarcomeric Protein Interactions in Drosophila
    Authors:  Océane Marescal, Frieder Schöck and Nicanor González-Morales, date: 04/05/2020, view: 652, Q&A: 0
    [Abstract] Protein-protein interactions in Drosophila myofibrils are essential for their function and formation. Bimolecular Fluorescence Complementation (BiFC) is an effective method for studying protein interactions and localization. BiFC relies on the reconstitution of a monomeric fluorescent protein from two half-fragments when in proximity. Two ...
    Super-resolution Microscopy-based Bimolecular Fluorescence Complementation to Study Protein Complex Assembly and Co-localization
    Authors:  Jingjing Chen, Zulin Yu, Jay R. Unruh, Brian D. Slaughter and Sue L. Jaspersen, date: 02/20/2020, view: 1285, Q&A: 0
    [Abstract] Numerous experimental approaches exist to study interactions between two subunits of a large macromolecular complex. However, most methods do not provide spatial and temporal information about binding, which are critical for dissecting the mechanism of assembly of nanosized complexes in vivo. While recent advances in super-resolution ...
    Assessing Self-interaction of Mammalian Nuclear Proteins by Co-immunoprecipitation
    Authors:  Claudia Cattoglio, Iryna Pustova, Xavier Darzacq, Robert Tjian and Anders S. Hansen, date: 02/20/2020, view: 1319, Q&A: 0
    [Abstract] Protein-protein interactions constitute the molecular foundations of virtually all biological processes. Co-immunoprecipitation (CoIP) experiments are probably the most widely used method to probe both heterotypic and homotypic protein-protein interactions. Recent advances in super-resolution microscopy have revealed that several nuclear proteins ...
    Surface Plasmon Resonance Analysis of the Protein-protein Binding Specificity Using Autolab ESPIRIT
    Authors:  Pragyan Parimita Rath, Gaurav Anand and Shalini Agarwal, date: 02/20/2020, view: 1199, Q&A: 0
    [Abstract] Direct protein-protein interactions are known to regulate a wide range of cellular activities. To understand these contacts one can employ various experimental methods like Dynamic Light Scattering (DLS), Fluorescence Resonance Energy Transfer (FRET), Isothermal titration calorimetry (ITC), Chemical crosslinking, Co-immunoprecipitation (Co-IP), ...
    Identification of Heteroreceptors Complexes and Signal Transduction Events Using Bioluminescence Resonance Energy Transfer (BRET)
    Authors:  Irene Reyes-Resina, Jasmina Jiménez, Gemma Navarro and Rafael Franco, date: 10/05/2019, view: 1336, Q&A: 0
    [Abstract] Detecting protein-protein interactions by co-immunoprecipitation provided a major advancement in the immunology research field. In the G-protein-coupled receptors (GPCRs) research field, colocalization and co-immunoprecipitation were used to detect interactions, but doubts arose due to specificity of the antibodies (monoclonal in the case of ...



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