Biochemistry

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    Protocols in Current Issue
    Purification of Globular Actin from Rabbit Muscle and Pyrene Fluorescent Assays to Investigate Actin Dynamics in vitro
    Authors:  He Sun, Yuanyuan Luo and Yansong Miao, date: 12/05/2018, view: 85, Q&A: 0
    Pyrene Fluorescent Assay is established to monitor the dynamic actin nucleation, elongation, capping and disassembly in vitro. This technique provides an easy handle procedure and straightforward visual data analysis. By coupling actin ...
    An in vitro Microscopy-based Assay for Microtubule-binding and Microtubule-crosslinking by Budding Yeast Microtubule-associated Protein
    Authors:  Yili Zhu, Weimin Tan and Wei-Lih Lee, date: 12/05/2018, view: 219, Q&A: 0
    In this protocol, we describe a simple microscopy-based method to assess the interaction of a microtubule-associated protein (MAP) with microtubules. The interaction between MAP and microtubules is typically assessed by a co-sedimentation assay, ...
    Integrin Dependent RhoB Activation Assay Using Leukocytes
    Authors:  Janne Lehtonen, Katarzyna Potrzebowska, Malin Samuelsson and Lena Svensson, date: 12/05/2018, view: 60, Q&A: 0
    To be able to migrate, leukocyte needs to re-use its adhesion molecules to move forward. These adhesion molecules are called integrins and are intracellularly transported via endocytosis and exocytosis in order to translocate to a new site on the ...
    Flow Cytometry Assay for Recycling of LFA-1 in T-lymphocytes
    Authors:  Katarzyna Potrzebowska, Janne Lehtonen, Malin Samuelsson and Lena Svensson, date: 12/05/2018, view: 107, Q&A: 0
    To enable cells to move forward, cell surface integrins are internalized into an endosomal compartment and subsequently intracellularly transported to be re-exposed at a new site on the cell membrane. Leukocytes are the fastest migrating cell type ...
    In vitro Membrane Interaction and Liposome Fusion Assays Using Recombinant Hepatitis C Virus Envelope Protein E2
    In order to study the mechanism underlying the Hepatitis C Virus (HCV) fusion process we have performed assays using phospholipid liposomes and a truncated form of E2 protein, E2661 (amino acids 384-661 of the HCV polyprotein) lacking the ...
    Wheat Germ Agglutinin (WGA)-SDS-PAGE: A Novel Method for the Detection of O-GlcNAc-modified Proteins by Lectin Affinity Gel Electrophoresis
    Authors:  Ko Fujioka, Yuji Kubota and Mutsuhiro Takekawa, date: 12/05/2018, view: 119, Q&A: 0
    Diverse cytoplasmic and nuclear proteins dynamically change their molecular functions by O-linked β-N-acetylglucosamine (O-GlcNAc) modification on serine and/or threonine residues. Evaluation of the O-GlcNAcylation level of a specific protein, ...
    Purification of Globular Actin from Rabbit Muscle and Pyrene Fluorescent Assays to Investigate Actin Dynamics in vitro
    Authors:  He Sun, Yuanyuan Luo and Yansong Miao, date: 12/05/2018, view: 85, Q&A: 0
    [Abstract] Pyrene Fluorescent Assay is established to monitor the dynamic actin nucleation, elongation, capping and disassembly in vitro. This technique provides an easy handle procedure and straightforward visual data analysis. By coupling actin purification and polymerization assays in this protocol, the readers could quickly get the affordable ...
    An in vitro Microscopy-based Assay for Microtubule-binding and Microtubule-crosslinking by Budding Yeast Microtubule-associated Protein
    Authors:  Yili Zhu, Weimin Tan and Wei-Lih Lee, date: 12/05/2018, view: 219, Q&A: 0
    [Abstract] In this protocol, we describe a simple microscopy-based method to assess the interaction of a microtubule-associated protein (MAP) with microtubules. The interaction between MAP and microtubules is typically assessed by a co-sedimentation assay, which measures the amount of MAP that co-pellets with microtubules by centrifugation, followed by ...
    Integrin Dependent RhoB Activation Assay Using Leukocytes
    Authors:  Janne Lehtonen, Katarzyna Potrzebowska, Malin Samuelsson and Lena Svensson, date: 12/05/2018, view: 60, Q&A: 0
    [Abstract] To be able to migrate, leukocyte needs to re-use its adhesion molecules to move forward. These adhesion molecules are called integrins and are intracellularly transported via endocytosis and exocytosis in order to translocate to a new site on the cell membrane. The intracellular transportation is regulated by different small GTPases including ...
    Flow Cytometry Assay for Recycling of LFA-1 in T-lymphocytes
    Authors:  Katarzyna Potrzebowska, Janne Lehtonen, Malin Samuelsson and Lena Svensson, date: 12/05/2018, view: 107, Q&A: 0
    [Abstract] To enable cells to move forward, cell surface integrins are internalized into an endosomal compartment and subsequently intracellularly transported to be re-exposed at a new site on the cell membrane. Leukocytes are the fastest migrating cell type in the human body, which express the leukocyte-specific integrin LFA-1. Here, we describe a flow ...
    In vitro Membrane Interaction and Liposome Fusion Assays Using Recombinant Hepatitis C Virus Envelope Protein E2
    [Abstract] In order to study the mechanism underlying the Hepatitis C Virus (HCV) fusion process we have performed assays using phospholipid liposomes and a truncated form of E2 protein, E2661 (amino acids 384-661 of the HCV polyprotein) lacking the transmembrane region. E2661 has been previously generated by using the baculovirus ...
    Wheat Germ Agglutinin (WGA)-SDS-PAGE: A Novel Method for the Detection of O-GlcNAc-modified Proteins by Lectin Affinity Gel Electrophoresis
    Authors:  Ko Fujioka, Yuji Kubota and Mutsuhiro Takekawa, date: 12/05/2018, view: 119, Q&A: 0
    [Abstract] Diverse cytoplasmic and nuclear proteins dynamically change their molecular functions by O-linked β-N-acetylglucosamine (O-GlcNAc) modification on serine and/or threonine residues. Evaluation of the O-GlcNAcylation level of a specific protein, however, needs multiple and time-consuming steps if using conventional methods (e.g., ...
    Purification of Soluble Recombinant Human Tau Protein from Bacteria Using Double-tag Affinity Purification
    Authors:  Joseph McInnes, Lujia Zhou and Patrik Verstreken, date: 11/20/2018, view: 340, Q&A: 0
    [Abstract] Dysfunction of the microtubule-associated protein Tau (encoded by the MAPT gene) has been implicated in more than twenty neurodegenerative diseases, including Alzheimer’s. As such, the physiological and disease-relevant functions of Tau have garnered great interest in the research community. One barrier hampering investigations into the ...
    Relative Quantitation of Polymerized Actin in Suspension Cells by Flow Cytometry
    Authors:  Mallory R. Kakley, Katrina B. Velle and Lillian K. Fritz-Laylin, date: 11/20/2018, view: 358, Q&A: 0
    [Abstract] The amount of polymerized actin within a cell can vary widely due to natural processes and/or experimentally induced perturbations. We routinely use this protocol to measure relative polymerized actin content between cell populations by staining cells in suspension with fluorescent phalloidin, then measuring total cell fluorescence using flow ...
    Separation and Visualization of Low Abundant Ubiquitylated Forms
    Authors:  Ramona Schuster, Tânia Simões, Fabian den Brave and Mafalda Escobar-Henriques, date: 11/20/2018, view: 465, Q&A: 0
    [Abstract] In this protocol we describe the separation and visualization of ubiquitylated forms of the yeast mitofusin Fzo1 by Western blot. To this aim, we express HA-tagged Fzo1 in Saccharomyces cerevisiae, break the cells to extract a membrane-enriched fraction, solubilize the membranes using detergent and then specifically immunoprecipitate the ...
    Eicosanoid Isolation from Mouse Intestinal Tissue for ELISA
    Author:  Isabella Rauch, date: 11/05/2018, view: 288, Q&A: 0
    [Abstract] Activation of inflammasomes in peritoneal macrophages and intestinal epithelial cells (IEC) leads to the release of eicosanoids. To assess the amount of eicosanoids released by IEC, lipids need to be isolated from whole tissue previous to analysis by lipid mass spectrometry or ELISA. This protocol describes how to isolate lipids from intestinal ...



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