Biochemistry

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    Protocols in Current Issue
    Isolation of Tumor Cells Based on Their Distance from Blood Vessels
    Authors:  Saran Kumar, Husni Sharife, Tirzah Kreisel, Libat Bar-Lev, Myriam Grunewald and Eli Keshet, date: 05/20/2020, view: 512, Q&A: 0
    Differential exposure of tumor cells to microenvironmental cues greatly impacts cell phenotypes, raising a need for position based sorting of tumor cells amenable to multiple OMICs and functional analyses. One such key determinant of tumor ...
    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: 396, Q&A: 0
    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 ...
    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: 321, Q&A: 0
    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 ...
    Pancreatic Acinar Cell Preparation for Oxygen Consumption and Lactate Production Analysis
    Authors:  Jane A. Armstrong, Robert Sutton and David N. Criddle, date: 05/20/2020, view: 361, Q&A: 0
    Mitochondrial dysfunction is a principal feature of acute pancreatitis (AP) although the underlying mechanisms are still unclear. AP precipitants induce Ca2+-dependent formation of the mitochondrial permeability transition pore (MPTP) 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: 494, Q&A: 0
    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 ...
    Negative Ion Mode nanoLC-ESI-MS/MS Analyses of Permethylated Sulfated Glycans
    Authors:  Shin-Yi Yu, Chu-Wen Cheng and Kay-Hooi Khoo, date: 05/20/2020, view: 445, Q&A: 0
    We have developed enabling techniques for sulfoglycomics based on MALDI-MS mapping and MS/MS sequencing of permethylated sulfated glycans. We then extended further the analytical workflow to C18 reverse phase (RP)-nanoLC-nanoESI-MS/MS analyses of ...
    Permethylation and Microfractionation of Sulfated Glycans for MS Analysis
    Authors:  Shin-Yi Yu, Sergei Snovida and Kay-Hooi Khoo, date: 05/20/2020, view: 459, Q&A: 0
    Sulfated glycans are barely detectable in routine mass spectrometry (MS)-based glycomic analysis due to ion suppression by the significantly more abundant neutral glycans in the positive ion mode, and sialylated non-sulfated glycans in the negative ...
    Evaluation of the Efficiency of Genome Editing Tools by a Frameshift Fluorescence Protein Reporter
    Authors:  Balaji T. Moorthy, Akhilesh Kumar, Lauren X. Lotenfoe and Fangliang Zhang, date: 05/20/2020, view: 539, Q&A: 0
    In the last decade, genome editing has been the center of attention as a novel tool for mechanistic investigations and for potential clinical applications. Various genome editing tools like meganucleases, zinc finger nucleases (ZFNs), transcription ...
    Spatial Image Correlation Spectroscopy (ICS): A Technique for Average Size Determination of Subcellular Accumulated Structures from Fluorescence Microscopic Images
    Authors:  Akira Kitamura and Masakata Kinjo, date: 05/20/2020, view: 424, Q&A: 0
    Size determination of subcellular structures such as inclusion bodies (IBs) and granules from fluorescent images is important for identification and structural characterization. However, it is often time-consuming just for the comparison of the ...
    Isolation of Tumor Cells Based on Their Distance from Blood Vessels
    Authors:  Saran Kumar, Husni Sharife, Tirzah Kreisel, Libat Bar-Lev, Myriam Grunewald and Eli Keshet, date: 05/20/2020, view: 512, Q&A: 0
    [Abstract] Differential exposure of tumor cells to microenvironmental cues greatly impacts cell phenotypes, raising a need for position based sorting of tumor cells amenable to multiple OMICs and functional analyses. One such key determinant of tumor heterogeneity in solid tumors is its vasculature. Proximity to blood vessels (BVs) profoundly affects tumor ...
    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: 396, 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: 321, 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 ...
    Pancreatic Acinar Cell Preparation for Oxygen Consumption and Lactate Production Analysis
    Authors:  Jane A. Armstrong, Robert Sutton and David N. Criddle, date: 05/20/2020, view: 361, Q&A: 0
    [Abstract] Mitochondrial dysfunction is a principal feature of acute pancreatitis (AP) although the underlying mechanisms are still unclear. AP precipitants induce Ca2+-dependent formation of the mitochondrial permeability transition pore (MPTP) in pancreatic acinar cells (PACs), leading to ATP depletion and necrosis. Evaluations of mitochondrial ...
    In vitro Assay for Bacterial Membrane Protein Integration into Proteoliposomes
    Authors:  Hanako Nishikawa, Masaru Sasaki and Ken-ichi Nishiyama, date: 05/20/2020, view: 494, 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 ...
    Negative Ion Mode nanoLC-ESI-MS/MS Analyses of Permethylated Sulfated Glycans
    Authors:  Shin-Yi Yu, Chu-Wen Cheng and Kay-Hooi Khoo, date: 05/20/2020, view: 445, Q&A: 0
    [Abstract] We have developed enabling techniques for sulfoglycomics based on MALDI-MS mapping and MS/MS sequencing of permethylated sulfated glycans. We then extended further the analytical workflow to C18 reverse phase (RP)-nanoLC-nanoESI-MS/MS analyses of permethylated sulfated glycans in the negative ion mode. The advantages are that extra sulfates on ...
    Permethylation and Microfractionation of Sulfated Glycans for MS Analysis
    Authors:  Shin-Yi Yu, Sergei Snovida and Kay-Hooi Khoo, date: 05/20/2020, view: 459, Q&A: 0
    [Abstract] Sulfated glycans are barely detectable in routine mass spectrometry (MS)-based glycomic analysis due to ion suppression by the significantly more abundant neutral glycans in the positive ion mode, and sialylated non-sulfated glycans in the negative ion mode, respectively. Nevertheless, the negative charge imparted by sulfate can be advantageous ...
    Evaluation of the Efficiency of Genome Editing Tools by a Frameshift Fluorescence Protein Reporter
    Authors:  Balaji T. Moorthy, Akhilesh Kumar, Lauren X. Lotenfoe and Fangliang Zhang, date: 05/20/2020, view: 539, Q&A: 0
    [Abstract] In the last decade, genome editing has been the center of attention as a novel tool for mechanistic investigations and for potential clinical applications. Various genome editing tools like meganucleases, zinc finger nucleases (ZFNs), transcription activator-like effector-based nucleases (TALEN), and the clustered regularly interspaced short ...
    Spatial Image Correlation Spectroscopy (ICS): A Technique for Average Size Determination of Subcellular Accumulated Structures from Fluorescence Microscopic Images
    Authors:  Akira Kitamura and Masakata Kinjo, date: 05/20/2020, view: 424, Q&A: 0
    [Abstract] Size determination of subcellular structures such as inclusion bodies (IBs) and granules from fluorescent images is important for identification and structural characterization. However, it is often time-consuming just for the comparison of the average size of the structures. Here, we introduce a high-throughput procedure to represent the average ...
    Confocal and Super-resolution Imaging of RNA in Live Bacteria Using a Fluorogenic Silicon Rhodamine-binding Aptamer
    Authors:  Regina Wirth, Peng Gao, G. Ulrich Nienhaus, Murat Sunbul and Andres Jäschke, date: 05/05/2020, view: 915, Q&A: 0
    [Abstract] Genetically encoded light-up RNA aptamers have been shown to be promising tools for the visualization of RNAs in living cells, helping us to advance our understanding of the broad and complex life of RNA. Although a handful of light-up aptamers spanning the visible wavelength region have been developed, none of them have yet been reported to be ...



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