Protocols in Current Issue
    Cell-free Fluorescent Intra-Golgi Retrograde Vesicle Trafficking Assay
    Authors:  Nathanael P. Cottam and Daniel Ungar, date: 11/20/2017, view: 102, Q&A: 0
    [Abstract] Intra-Golgi retrograde vesicle transport is used to traffic and sort resident Golgi enzymes to their appropriate cisternal locations. An assay was established to investigate the molecular details of vesicle targeting in a cell-free system. Stable cell lines were generated in which the trans-Golgi enzyme galactosyltransferase (GalT) was ...
    γ-Secretase Epsilon-cleavage Assay
    [Abstract] γ-Secretase epsilon-cleavage assay is derived from the cell-based Tango assay (Kang et al., 2015), and is a fast and sensitive method to determine the initial cleavage of C99 by γ-secretase. In this protocol, we use HTL cells, which are HEK293 cells with a stably integrated luciferase reporter under the control of the bacterial tetO ...
    In vitro NLK Kinase Assay
    Authors:  Sungho Moon, Jiyoung Kim and Eek‐hoon Jho, date: 11/05/2017, view: 248, Q&A: 0
    [Abstract] This protocol provides step by step instructions to perform an in vitro kinase assay for nemo-like kinase. In addition, this protocol also describes an efficient method using mild lysis buffer for expression and purification of Glutathione S-transferase (GST) fusion proteins.
    Uptake Assays in Xenopus laevis Oocytes Using Liquid Chromatography-mass Spectrometry to Detect Transport Activity
    [Abstract] Xenopus laevis oocytes are a widely used model system for characterization of heterologously expressed secondary active transporters. Historically, researchers have relied on detecting transport activity by measuring accumulation of radiolabeled substrates by scintillation counting or of fluorescently labelled substrates by ...
    In vitro Assays for Eukaryotic Leading/Lagging Strand DNA Replication
    Authors:  Grant Schauer, Jeff Finkelstein and Mike O’Donnell, date: 09/20/2017, view: 824, Q&A: 0
    [Abstract] The eukaryotic replisome is a multiprotein complex that duplicates DNA. The replisome is sculpted to couple continuous leading strand synthesis with discontinuous lagging strand synthesis, primarily carried out by DNA polymerases ε and δ, respectively, along with helicases, polymerase α-primase, DNA sliding clamps, clamp loaders and many other ...
    Isolation and Detection of the Chlorophyll Catabolite Hydroxylating Activity from Capsicum annuum Chromoplasts
    Authors:  Mareike Hauenstein and Stefan Hörtensteiner, date: 09/20/2017, view: 724, Q&A: 0
    [Abstract] Hydroxylation of chlorophyll catabolites at the so-called C32 position (Hauenstein et al., 2016) is commonly found in all plant species analyzed to date. Here we describe an in vitro hydroxylation assay using Capsicum annuum chromoplast membranes as a source of the hydroxylating activity, which converts the ...
    Protease Activity Assay in Fly Intestines
    Authors:  Marie-Paule Nawrot-Esposito, Rihab Loudhaief and Armel Gallet, date: 09/20/2017, view: 728, Q&A: 0
    [Abstract] The intestine is a central organ required for the digestion of food, the absorption of nutrients and for fighting against aggressors ingested along with the food. Impairment of gut physiology following mucosal damages impacts its digestive capacities that consequently will affect growth, wellbeing or even survival of the individual. Hence, the ...
    Superoxide Dismutase (SOD) and Catalase (CAT) Activity Assay Protocols for Caenorhabditis elegans
    Authors:  Jing Zhang, Rui Chen, Zhenyang Yu and Lili Xue, date: 08/20/2017, view: 867, Q&A: 0
    [Abstract] Assays for superoxide dismutase (SOD) and catalase (CAT) activities are widely employed to indicate antioxidant responses underlying the toxic effects of test chemicals. Yet, earlier studies mainly described the procedures as performed according to manufacturer’s instructions without modifications that are specific to any organisms. The present ...
    Liposome Disruption Assay to Examine Lytic Properties of Biomolecules
    Authors:  John R. Jimah, Paul H. Schlesinger and Niraj H. Tolia, date: 08/05/2017, view: 1065, Q&A: 0
    [Abstract] Proteins may have three dimensional structural or amino acid features that suggest a role in targeting and disrupting lipids within cell membranes. It is often necessary to experimentally investigate if these proteins and biomolecules are able to disrupt membranes in order to conclusively characterize the function of these biomolecules. Here, we ...
    Membrane Lipid Screen to Identify Molecular Targets of Biomolecules
    Authors:  John R. Jimah, Paul H. Schlesinger and Niraj H. Tolia, date: 08/05/2017, view: 968, Q&A: 0
    [Abstract] Proteins that bind to and disrupt cell membranes may target specific phospholipids. Here we describe a protocol to identify the lipid targets of proteins and biomolecules. First, we describe a screen to identify lipids in membranes that are specifically bound by the biomolecule of interest. Second, we describe a method for determining if the ...