Biochemistry

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    Protocols in Current Issue
    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: 186, Q&A: 0
    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 ...
    Estimating Cellular Abundances of Halo-tagged Proteins in Live Mammalian Cells by Flow Cytometry
    Authors:  Claudia Cattoglio, Xavier Darzacq, Robert Tjian and Anders Sejr Hansen, date: 02/20/2020, view: 256, Q&A: 0
    Accurate abundance measurements of cellular proteins are required to achieve a quantitative and predictive understanding of any biological process inside the cell. Existing methods to determine absolute protein abundances are labor-intensive and/or ...
    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: 235, Q&A: 0
    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 ...
    Construction of Antisense RNA-mediated Gene Knock-down Strains in the Cyanobacterium Anabaena sp. PCC 7120
    Authors:  Amit Srivastava, Anand Ballal, Karl Forchhammer and Anil Kumar Tripathi, date: 02/20/2020, view: 58, Q&A: 0
    Anabaena sp. PCC 7120 (hereafter Anabaena) is a model cyanobacterium to study nitrogen fixation, cellular differentiation and several other key biological functions that are analogous in plants. As with any other organism, many ...
    Photoactivable Cholesterol as a Tool to Study Interaction of Influenza Virus Hemagglutinin with Cholesterol
    Authors:  Bodan Hu, Mohamed Rasheed Gadalla, Christoph Thiele and Michael Veit, date: 02/20/2020, view: 53, Q&A: 0
    Non-covalent binding of cholesterol to the transmembrane region of proteins affect their functionalities, but methods to prove such an interaction are rare. We describe our protocol to label the hemagglutinin (HA) of Influenza virus with a ...
    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: 186, 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 ...
    Estimating Cellular Abundances of Halo-tagged Proteins in Live Mammalian Cells by Flow Cytometry
    Authors:  Claudia Cattoglio, Xavier Darzacq, Robert Tjian and Anders Sejr Hansen, date: 02/20/2020, view: 256, Q&A: 0
    [Abstract] Accurate abundance measurements of cellular proteins are required to achieve a quantitative and predictive understanding of any biological process inside the cell. Existing methods to determine absolute protein abundances are labor-intensive and/or require sophisticated experimental and computational infrastructure (e.g., fluorescence ...
    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: 235, 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 ...
    Construction of Antisense RNA-mediated Gene Knock-down Strains in the Cyanobacterium Anabaena sp. PCC 7120
    Authors:  Amit Srivastava, Anand Ballal, Karl Forchhammer and Anil Kumar Tripathi, date: 02/20/2020, view: 58, Q&A: 0
    [Abstract] Anabaena sp. PCC 7120 (hereafter Anabaena) is a model cyanobacterium to study nitrogen fixation, cellular differentiation and several other key biological functions that are analogous in plants. As with any other organism, many genes in Anabaena encode an essential life function and hence cannot be deleted, causing a ...
    Photoactivable Cholesterol as a Tool to Study Interaction of Influenza Virus Hemagglutinin with Cholesterol
    Authors:  Bodan Hu, Mohamed Rasheed Gadalla, Christoph Thiele and Michael Veit, date: 02/20/2020, view: 53, Q&A: 0
    [Abstract] Non-covalent binding of cholesterol to the transmembrane region of proteins affect their functionalities, but methods to prove such an interaction are rare. We describe our protocol to label the hemagglutinin (HA) of Influenza virus with a cholesterol derivative in living cells or with immunoprecipitated protein. We synthesized a “clickable” ...
    Expression and Purification of Adeno-associated Virus Virus-like Particles in a Baculovirus System and AAVR Ectodomain Constructs in E. coli
    [Abstract] Adeno-associated virus (AAV) is a promising gene therapy vector and the biophysical characterization of its interactions with host proteins is a critical foundation for engineering tissue targeting and immune escape. Presented here are protocols for the production of: (a) the outer protein shells (virus-like particles or VLPs) for serotype 2 ...
    Evaluating Whole Blood Clotting in vitro on Biomaterial Surfaces
    Authors:  Roberta M Sabino and Ketul C Popat, date: 02/05/2020, view: 297, Q&A: 0
    [Abstract] Biomaterial-associated thrombosis is still a major concern for blood-contacting implants. After the medical device is implanted and comes in contact with blood, several complex reactions occur, which may lead to thrombus formation and failure of the device. Therefore, it is essential to evaluate the biomaterial interaction with the whole blood. ...
    Identification of Target Protein for Bio-active Small Molecule Using Photo-cross Linked Beads and MALDI-TOF Mass Spectrometry
    [Abstract] Development of methods for protein identification is one of the important aspects of proteomics. Here, we report a protocol for the preparation of compound conjugated beads by photo-crosslinking, affinity purification, gel electrophoresis, and highly sensitive mass spectrometric assay for drug-target identification. Although there are several ...
    Measurement of ATPase Activity of Valosin-containing Protein/p97
    [Abstract] Valosin-containing protein (VCP; also known as p97) is a type II ATPase regulating several cellular processes. Using proteomic techniques, we identified a chemical compound that binds to the D1 ATPase domain of VCP. The protocol described here was to study the effect of the compound on ATPase activity in vitro of purified VCP protein. ...
    Live Mitochondrial or Cytosolic Calcium Imaging Using Genetically-encoded Cameleon Indicator in Mammalian Cells
    Authors:  Elisa Greotti and Tullio Pozzan, date: 02/05/2020, view: 264, Q&A: 0
    [Abstract] Calcium (Ca2+) imaging aims at investigating the dynamic changes in live cells of its concentration ([Ca2+]) in different pathophysiological conditions. Ca2+ is an ubiquitous and versatile intracellular signal that modulates a large variety of cellular functions thanks to a cell type-specific toolkit and a complex ...



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