Molecular Biology

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    Protocols in Current Issue
    Purification of Protein-complexes from the Cyanobacterium Synechocystis sp. PCC 6803 Using FLAG-affinity Chromatography
    Authors:  Minna M. Koskela, Petra Skotnicová, Éva Kiss and Roman Sobotka, date: 05/20/2020, view: 243, Q&A: 0
    [Abstract] Exploring the structure and function of protein complexes requires their isolation in the native state–a task that is made challenging when studying labile and/or low abundant complexes. The difficulties in preparing membrane-protein complexes are especially notorious. The cyanobacterium Synechocystis sp. PCC 6803 is a widely used model ...
    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: 206, 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: 218, 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 ...
    In vitro Assay for Bacterial Membrane Protein Integration into Proteoliposomes
    Authors:  Hanako Nishikawa, Masaru Sasaki and Ken-ichi Nishiyama, date: 05/20/2020, view: 232, Q&A: 0
    [Abstract] It is important to experimentally determine how membrane proteins are integrated into biomembranes to unveil the roles of the integration factors, and to understand the functions and structures of membrane proteins. We have developed a reconstitution system for membrane protein integration in E. coli using purified factors, in which the ...
    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: 287, 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 ...
    Enzymatic Construction of Protein Polymer/Polyprotein Using OaAEP1 and TEV Protease
    Authors:  Yibing Deng, Shengchao Shi, Bin Zheng, Tao Wu and Peng Zheng, date: 04/20/2020, view: 763, Q&A: 0
    [Abstract] The development of chemical and biological coupling technologies in recent years has made possible of protein polymers engineering. We have developed an enzymatic method for building polyproteins using a protein ligase OaAEP1 (asparagine endopeptidase 1) and protease TEV (tobacco etching virus). Using a mobile TEV protease site compatible with the ...
    Isolation and Characterization of Exosomes from Mouse Feces
    Authors:  Chunhua Yang, Mingzhen Zhang, Junsik Sung, Lixin Wang, Yunjin Jung and Didier Merlin, date: 04/20/2020, view: 592, Q&A: 0
    [Abstract] Exosomes secreted by colonic epithelial cells are present in feces and contain valuable epigenetic information, such as miRNAs, proteins, and metabolites. An in-depth study of this information is conducive to the diagnosis or treatment of relevant diseases. A crucial prerequisite of such a study is to establish an efficient isolation method, ...
    Dual Fluorescence Cytometry Assay to Assess Cellular Protein Levels
    Authors:  Ashley M Ngo, Ruofan Wang and Andreas S Puschnik, date: 04/20/2020, view: 735, Q&A: 0
    [Abstract] Expression levels of cellular proteins can be affected by various perturbations, such as genetic knockout of interactors, drug treatments or cell stress. To specifically measure the effects on protein levels post-synthesis under different experimental conditions, it is important to compensate for transcriptional and other upstream changes. Here, ...
    An in vitro Assay to Screen for Substrates of PKA Using Phospho-motif-specific Antibodies
    Authors:  David M. Gau and Partha Roy, date: 04/20/2020, view: 373, Q&A: 0
    [Abstract] Kinases function as regulators of many cellular processes such as cell migration. These enzymes typically phosphorylate target motif sequences. Mass spec or phospho-specific antibody detection can be used to determine whether a kinase can phosphorylate proteins of interest, however, mass spec can be expensive and phospho-antibodies for the protein ...
    Auxin-mediated Protein Degradation in Caenorhabditis elegans
    Authors:  Michael A. Q. Martinez and David Q. Matus, date: 04/20/2020, view: 691, Q&A: 0
    [Abstract] The auxin-inducible degron (AID) technology was recently adapted for use in the nematode Caenorhabditis elegans. Rapid degradation of C. elegans proteins tagged with an AID is mediated by a plant-specific F-box protein, transport inhibitor response 1 (TIR1), and occurs only in the presence of the phytohormone auxin. The first ...



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