Cell Biology

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    Protocols in Current Issue
    Quantification of Protein Kinase A (PKA) Activity by An in vitro Radioactive Assay Using the Mouse Sperm Derived Enzyme
    Authors:  Cintia Stival, Carolina Baro Graf, Pablo E. Visconti and Dario Krapf, date: 06/20/2020, view: 572, Q&A: 0
    [Abstract] In order to acquire fertilizing potential, mammalian sperm must undergo a process known as capacitation, which relies on the early activation of Protein Kinase A (PKA). Frequently, PKA activity is assessed in whole-cell experiments by analyzing the phosphorylation status of its substrates in a western-blot. This technique ...
    In vitro Crosslinking Reactions and Substrate Incorporation Assays for The Identification of Transglutaminase-2 Protein Substrates
    Authors:  William L. Willis, Abigail Foster, Caitlin Henry, Lai Chu Wu and Wael Jarjour, date: 06/20/2020, view: 465, Q&A: 0
    [Abstract] Transglutaminase (TG2) catalyzes protein crosslinking between glutamyl and lysyl residues. Catalytic activity occurs via a transamidation mechanism resulting in the formation of isopeptide bonds. Since TG2-mediated transamidation is of mechanistic importance for a number of biological processes, assays that enable rapid and efficient ...
    Preparation of HeLa Total Membranes and Assay of Lipid-inhibition of Serine Palmitoyltransferase Activity
    [Abstract] Serine palmitoyltranferase (SPT) is a pyridoxal 5′ phosphate (PLP)-dependent enzyme that catalyzes the first and rate-limiting step of de novo synthesis of sphingolipids. SPT activity is homeostatically regulated in response to increased levels of sphingolipids. This homeostatic regulation of SPT is mediated through small ER membrane ...
    A Sensitive Coupled Enzyme Assay for Measuring Kinase and ATPase Kinetics Using ADP-Specific Hexokinase
    Authors:  Ciaran R. McFarlane and James W. Murray, date: 05/05/2020, view: 635, Q&A: 0
    [Abstract] Kinases and ATPases perform essential biological functions in metabolism and regulation. Activity of these enzymes is commonly measured by coupling ATP consumption to the synthesis of a detectable product. For most assay systems the ATP concentration during the reaction is unknown, compromising the precision of the assay.

    Using the ...
    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: 556, 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 ...
    Fibrin Breakdown Assay
    Authors:  Jiayue Ling, Connor M. Blair and George S. Baillie, date: 04/20/2020, view: 979, Q&A: 0
    [Abstract] Fibrinolysis is an integral part of the matrix remodeling process that contributes to tissue repair. Fibrin clots are broken down during fibrinolysis in a controlled process. Fibrin degradation products (FDPs) have also been shown to have a role in the regulation of cell growth and are implicated in various vascular diseases. This protocol was ...
    Flow Cytometry Measurement of Glucocerebrosidase Activity in Human Monocytes
    Authors:  Laura P. Hughes, Glenda M. Halliday and Nicolas Dzamko, date: 04/05/2020, view: 630, Q&A: 0
    [Abstract] Glucocerebrosidase (GCase) is an important enzyme for the metabolism of glycolipids. GCase enzyme deficiency is implicated in human disease and the efficient measurement of GCase activity is important for evaluating the efficacy of therapeutics targeting this enzyme. Existing approaches to measure GCase activity include whole blood mass ...
    Measurement of Acid Ecto-phosphatase Activity in Live Leishmania donovani Parasites
    Authors:  Amalia Papadaki and Haralabia Boleti, date: 10/05/2019, view: 1294, Q&A: 0
    [Abstract] Acid ecto-phosphatases are enzymes that hydrolyze phosphomonoesters in the acidic pH range with their active sites facing the extacellular medium. Their activities can be measured in living cells. In bacteria and protozoan pathogens, acid ecto-phosphatases have been associated with the survival of intracellular pathogens within phagocytes through ...



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